Cyclic peptides targeting alpha-4-beta-7 integrin

ABSTRACT

There is described herein antagonists of α4β7 integrin, and more particularly to cyclic peptide antagonists. Accordingly, there is described herein a compound of formula (I) wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7  and R 8  are various substituents; stereocentres 1*, 2* and 3* are each independently selected from R and S; n is 1, 2, 3, or 4 and where n is 2-4, Z is an amino terminus of an amino acid; —C═O— adjacent L is the carboxy terminus of an amino acid; and L along with Z and —C═O— is a peptide.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No. 62/254003 filed on Nov. 11, 2015, incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to antagonists of α4β7 integrin, and more particularly to cyclic peptide antagonists.

BACKGROUND OF THE INVENTION

Integrins are transmembrane receptors that are the bridges for cell-cell and cell-extracellular matrix (ECM) interactions. When triggered, integrins trigger chemical pathways to the interior (signal transduction), such as the chemical composition and mechanical status of the ECM.

Integrins are obligate heterodimers, having two different chains: the α (alpha) and β (beta) subunits.

The α4β7 integrin is expressed on lymphocytes and is responsible for T-cell homing into gut-associated lymphoid tissues through its binding to mucosal addressin cell adhesion molecule (MAdCAM), which is present on high endothelial venules of mucosal lymphoid organs.

Inhibitors of specific integrin-ligand interactions have been shown effective as anti-inflammatory agents for the treatment of various autoimmune diseases. For example, monoclonal antibodies displaying high binding affinity for α4β7 have displayed therapeutic benefits for gastrointestinal auto-inflammatory/autoimmune diseases, such as Crohn's disease, and ulcerative colitis.

There is a need to develop improved α4β7 antagonists to prevent or treat inflammatory conidtions and/or autoimmune diseases.

Certain methods of making cyclic peptides (nacellins) are described in Applicant's PCT Publication No. WO 2010/105363.

SUMMARY OF THE INVENTION

In an aspect, there is provided, a compound of formula (I):

wherein

R¹ is H; lower alkyl; aryl; heteroaryl; alkenyl; or heterocycle; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents;

R² and R³ are each independently an amino acid chain of a proteinogenic or a non-proteinogenic alpha-amino acid,

-   -   provided that R² and R³ may be covalently linked to each other         to form a ring;

R⁴ and R⁵ are each independently H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; acids of the formula —C(O)OH; esters of the formula —C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula —C(O)NR**R***, wherein R** and R*** are independently selected from H, alkyl and aryl; —CH₂C(O)R, wherein R is selected from —OH, lower alkyl, aryl, -loweralkyl-aryl, or —NRaRb, where Ra and Rb are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; or —C(O)Rc, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-ORd, wherein Rd is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents;

-   -   provided that R² or R³ can be covalently linked to R¹ to form a         cyclic secondary amine, and/or to R⁴ or R⁵ to form a ring, R⁴         and R⁵ may also be covalently linked to each other to form a         ring;

R⁶ is H, lower alkyl, benzyl, alkenyl, lower alkyloxy; aryl; heteroaryl; heterocycle; —C(O)R****, wherein R**** is independently selected from alkyl, aryl, heteroaryl, amino, aminoalkyl, aminoaryl, aminoheteroaryl, alkoxy, aryloxy, heteroaryloxy; —CH₂C(O)R; or —C(O)Rc; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents,

-   -   or along with R⁷ or R⁸, a cyclic side chain of a proteinogenic         or a non-proteinogenic amino acid having, the N-terminus thereof         being the N—R⁶, wherein the proteinogenic or a non-proteinogenic         amino acid can be substituted with a suitable substituent;

R⁷ and R⁸ are independently selected from the amino acid side chains of a proteinogenic or a non-proteinogenic alpha-amino acid having the N-terminus thereof being the N—R⁶, or may form a cyclic side chain with R⁶;

stereocentres 1*, 2* and 3* are each independently selected from R and S;

n is 1, 2, 3, or 4 and where n is 2-4, each R⁷ and each R⁸ are independent of each other; and

wherein Z is an amino terminus of an amino acid; —C═O— adjacent L is the carboxy terminus of an amino acid; and L along with Z and —C═O— is a peptide having the following formula:

-   -   wherein X^(y) and X^(z) are each independently a proteinogenic         or non-proteinogenic amino acid;     -   X¹ is Leucine or tert-butyl-Ala;     -   X² is Asp; and     -   X³ is any amino acid listed under column X³ of Table 1B.

In an aspect, there is provided, a pharmaceutical composition comprising a compound described herein along with the pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for any one of oral delivery, topical delivery and parenteral delivery.

In an aspect, there is provided, a method of treating inflammation or an autoimmune disease in a patient, comprising administering to the patient a therapeutically effective amount of the compound described herein. Preferably the inflammation or an autoimmune disease is gastrointestinal.

In an aspect, there is provided, a method for treating a condition in a patient associated with a biological function of an α4β7 integrin, the method comprising administering to the patient a therapeutically effective amount of the compound described herein.

In an aspect, there is provided, a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the compound described herein, wherein the disease or condition is a local or systemic infection of a virus or retrovirus.

In an aspect, there is provided, a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the compound described herein, wherein the hepatitis A, B or C, hepatic encephalopathy, non-alcoholic steatohepatitis, cirrhosis, variceal bleeding, hemochromatosis, Wilson disease, tyrosinemia, alpha-1-antitrypsin deficiency, glycogen storage disease, hepatocellular carcinoma, liver cancer, primary biliary cholangitis, primary sclerosing cholangitis, primary biliary sclerosis, biliary tract disease, autoimmune hepatitis, or graft-versus-host disease.

BRIEF DESCRIPTION OF FIGURES AND TABLES

These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings and tables wherein:

FIG. 1 shows representative compounds of the present application, namely from the following classes, 18-membered ring, 21-membered ring, 21-membered ring (non-canonical, i.e. having a delta amino acid), 22-membered ring, and 24-membered ring.

FIG. 2 shows a representative 18-membered ring compound along with variations made at certain positions with corresponding α4β7 integrin ELISA IC50 binding values associated with those variations.

FIG. 3 shows a representative 21-membered ring compound along with variations made at certain positions with corresponding α4β7 integrin ELISA IC50 binding values associated with those variations.

FIG. 4 shows a representative 21-membered ring (non-canonical, i.e. having a delta amino acid) compound along with variations made at certain positions with corresponding α4β7 integrin ELISA IC50 binding values associated with those variations.

FIG. 5 shows a representative 22-membered ring compound along with variations made at certain positions with corresponding α4β7 integrin ELISA IC50 binding values associated with those variations.

FIG. 6 shows results of T lymphocyte trafficking studies (from peripheral blood to mesenteric lymph nodes) following single doses of various compounds.

FIG. 7 shows the pharmacokinetic profile for a test article (ET1792) via one and two oral doses in naïve mice.

FIG. 8 shows the pharmacokinetic profile for a test article (ET1792) after a single oral dose.

FIG. 9 shows the exposure of ET2451 in the liver of naïve mice that have received the test compound as a single oral or intravenous dose.

FIG. 10 shows the disease activity index score for the various treatment groups at day 5 and day 8 following the initiation of dextran sulfate sodium treatment in mice.

FIG. 11 shows data from ex vivo assessments of the colon taken from mice exposed to DSS and treated with various test compounds.

FIG. 12 shows further detail on the colon injury following DSS exposure and test nacellin treatment in mice.

FIG. 13 shows the outcome of FACS analyses of T lymphocyte content in peripheral lymph nodes, mesenteric lymph nodes and peripheral blood taken from mice exposed to DSS irritant and treated for three days with various test nacellins or control.

FIG. 14 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 9.

FIG. 15 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 10.

FIG. 16 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 11.

FIG. 17 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 12.

FIG. 18 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 14.

FIG. 19 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 15.

FIG. 20 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 34.

FIG. 21 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 40.

FIG. 22 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 41.

FIG. 23 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 42.

FIG. 24 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 43.

FIG. 25 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 45.

FIG. 26 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 47.

FIG. 27 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 50.

FIG. 28 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 100.

FIG. 29 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 101.

FIG. 30 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 103

FIG. 31 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 104.

FIG. 32 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 106.

FIG. 33 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 107.

FIG. 34 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 108.

FIG. 35 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 109.

FIG. 36 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 110.

FIG. 37 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 111.

FIG. 38 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 112.

FIG. 39 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 123.

FIG. 40 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 126.

FIG. 41 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 127.

FIG. 42 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 128.

FIG. 43 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 129.

FIG. 44 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 229.

FIG. 45 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 230.

FIG. 46 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 266.

FIG. 47 is an ¹H NMR at 25° C. and ¹H-¹H TOCSY NMR at 25° C. of Compound No. 269.

Table 1 shows compounds exhibiting α4β7 integrin affinity, selectivity and/or activity; and specifically with respect to these compounds: (A) the structure of the linker portion; (B) the structure of the peptide portion; and (C) the affinity, selectivity and activity values.

To aid reading of the table, the following is noted:

Table 1A:

-   -   If R2 is H and R3 is CH3, the carbon atom bearing R2 and R3 has         S-configuration.     -   If R2 is CH3 and R3 is H, the carbon atom bearing R2 and R3 has         R-configuration.     -   If R2 is H and R3 is CH2-S-Ph, the carbon atom bearing R2 and R3         has S-configuration.     -   If R4 is H and R5 is C(O)—NH-tert-Butyl, the carbon atom bearing         R4 and R5 has S-configuration.     -   If R4 is C(0)-NH-tert-Butyl and R5 is H, the carbon atom bearing         R4 and R5 has R-configuration.     -   If R1 and R2 are both Pro-, the R1 and R2 substituents are         covalently bound and form the pyrrolidine ring of Pro.

Table 1B

-   -   If R6 and R7 are both Pro, the R6 and R7 substituents are         covalently bound and form the pyrrolidine ring of Pro.     -   If R6 and R8 are both dPro, the R6 and R8 substituents are         covalently bound and form the pyrrolidine ring of dPro.     -   If R6 and R7 are both [(4S)-fluoro-Pro], the R6 and R7         substituents are covalently bound and form the pyrrolidine ring         of [(4S)-fluoro-Pro].     -   If R7 is Nva and R8 is H, the carbon atom bearing R7 and R8 has         S-configuration.     -   If R6 and R7 are both Hyp, the R6 and R7 substituents are         covalently bound and form the pyrrolidine ring of Hyp.     -   If no entry exists under column Xz, the residue is absent.

Table 1C

-   -   If no entry exists under any of the columns, no data was         collected.

Table 2 shows compounds exhibiting less α4β7 integrin affinity, selectivity and/or activity; and specifically with respect to these compounds: (A) the structure of the linker portion; (B) the structure of the peptide portion; and (C) the affinity, selectivity and activity values.

To aid reading of the table, the following is noted:

Table 2A

-   -   If R2 is H and R3 is CH3, the carbon atom bearing R2 and R3 has         S-configuration.     -   If R2 is CH3 and R3 is H, the carbon atom bearing R2 and R3 has         R-configuration.     -   If R4 is H and R5 is C(0)-NH-tert-Butyl, the carbon atom bearing         R4 and R5 has S-configuration.     -   If R4 is C(0)-NH-tert-Butyl and R5 is H, the carbon atom bearing         R4 and R5 has R-configuration.     -   If R1 and R2 are both Pro-, the R1 and R2 substituents are         covalently bound and form the pyrrolidine ring of Pro.

Table 2B

-   -   If R6 and R7 are both Pro, the R6 and R7 substituents are         covalently bound and form the pyrrolidine ring of Pro.     -   If R6 and R8 are both dPro, the R6 and R8 substituents are         covalently bound and form the pyrrolidine ring of dPro.     -   If no entry exists under column Xz, the residue is absent.

Table 2C

-   -   If no entry exists under any of the columns, no data was         collected.

Table S1 is a correspondence table linking the compounds described herein with the synthesis protocols outlined in the methods and materials.

DETAILED DESCRIPTION

In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.

In an aspect, there is provided, a compound of formula (I):

wherein

R¹ is H; lower alkyl; aryl; heteroaryl; alkenyl; or heterocycle; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents;

R² and R³ are each independently an amino acid chain of a proteinogenic or a non-proteinogenic alpha-amino acid,

-   -   provided that R² and R³ may be covalently linked to each other         to form a ring;

R⁴ and R⁵ are each independently H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; acids of the formula —C(O)OH; esters of the formula —C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula —C(O)NR**R***, wherein R** and R*** are independently selected from H, alkyl and aryl; —CH₂C(O)R, wherein R is selected from —OH, lower alkyl, aryl, -loweralkyl-aryl, or —NRaRb, where Ra and Rb are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; or —C(O)Rc, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-ORd, wherein Rd is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents;

-   -   provided that R² or R³ can be covalently linked to R¹ to form a         cyclic secondary amine, and/or to R⁴ or R⁵ to form a ring, R⁴         and R⁵ may also be covalently linked to each other to form a         ring;

R⁶ is H, lower alkyl, benzyl, alkenyl, lower alkyloxy; aryl; heteroaryl; heterocycle; —C(O)R****, wherein R**** is independently selected from alkyl, aryl, heteroaryl, amino, aminoalkyl, aminoaryl, aminoheteroaryl, alkoxy, aryloxy, heteroaryloxy; —CH₂C(O)R; or —C(O)Rc; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents,

-   -   or along with R⁷ or R⁸, a cyclic side chain of a proteinogenic         or a non-proteinogenic amino acid having, the N-terminus thereof         being the N—R⁶, wherein the proteinogenic or a non-proteinogenic         amino acid can be substituted with a suitable substituent;

R⁷ and R⁸ are independently selected from the amino acid side chains of a proteinogenic or a non-proteinogenic alpha-amino acid having the N-terminus thereof being the N—R⁶, or may form a cyclic side chain with R⁶;

stereocentres 1*, 2* and 3* are each independently selected from R and S;

n is 1, 2, 3, or 4 and where n is 2-4, each R⁷ and each R⁸ are independent of each other; and

wherein Z is an amino terminus of an amino acid; —C═O— adjacent L is the carboxy terminus of an amino acid; and L along with Z and —C═O— is a peptide having the following formula:

-   -   wherein X^(y) and X^(z) are each independently a proteinogenic         or non-proteinogenic amino acid;     -   X¹ is Leucine or tert-butyl-Ala;     -   X² is Asp; and     -   X³ is any amino acid listed under column X³ of Table 1B.

The compounds shown in Tables 1A, 1B and 1C exhibit antagonistic activity against α4β7 integrin and having selectivity over α4β1 integrin. A person skilled in the art would expect that substituents R¹-R⁸ and amino acids X^(y), X^(z), X¹, X², and X³ outlined in -Tables 1A and 1B with respect to different compounds could be combined in any manner and would likely result in a compound that would exhibit α4β7 integrin activity and selectivity.

As used herein, the term “amino acid” refers to molecules containing an amine group, a carboxylic acid group and a side chain that varies. Amino acid is meant to include not only the twenty amino acids commonly found in proteins but also non-standard amino acids and unnatural amino acid derivatives known to those of skill in the art, and therefore includes, but is not limited to, alpha, beta and gamma amino acids. Peptides are polymers of at least two amino acids and may include standard, non-standard, and unnatural amino acids. A peptide is a polymer of two or more amino acids.

The following abbreviations are used herein:

Abbreviation Description 1,2-cis-ACHC cis-2-aminocyclohexanecarboxylic acid 1,2-trans-ACHC trans-2-aminocyclohexanecarboxylic acid 1Nal 1-napthylalanine 2Abz anthranilic acid, 2-aminobenzoic acid 2Igl 2-indanylglycine 2Nal 2-napthylalanine Abu 2-aminobutyric acid Aic aminoindan-2-carboxylic acid allolle allo-sioleucine, (2S,3R)-2-amino-3-methylpentanoic acid alloThr allo-threonine, (2S,3S)-2-amino-3-hydroxybutyric acid alphaMePhe α-methyl-phenylalanine, (S)-(−)-2-amino-2-methyl-3-phenylpropionic acid Asp(ethyl ester) aspartic acid β-ethyl ester Atc 2-aminotetraline-2-carboxylic acid Aze azetidine-2-carboxylic acid BHT butylated hydroxytoluene Bip biphenylalanine C10 sebacic acid C12 dodecanedioic C7 pimelic acid C8 suberic acid C9 azelaic acid Cha β-cyclohexyl alanine, (S)-2-amino-3-cyclohexylpropionic acid Chg cyclohexyl glycine cis-dhyp cis-D-4-Hydroxyproline, (2R,4R)-4-Hydroxypyrrolidine-2-carboxylic acid cycloLeu cyclo leucine, 1-Aminocyclopentane-1-carboxylic acid cyclopropylAla β-cyclopropyl alanine, (S)-2-amino-3-cyclopropyl-propionic acid d2Igl 2-indanyl-D-glycine Dap(Cbz) Nβ-Z-2,3-diaminopropionic acid DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DEPBT 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one dHyp trans-D-4-hydroxyproline, (2R,4S)-4-hydroxypyrrolidine-2-carboxylic acid DIAD diisopropyl azodicarboxylate DIG diglycolic acid DIPEA N,N-diisopropylethylamine DMAP 4-(Dimethylamino)pyridine dMeArg N-methyl-D-arginine dMebetaHomoLys N-methyl-D-β-homoLys dMeLys N-methyl-D-Lysine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide dNle D-norleucine dOrn D-ornithine dOrn(dimethyl) Nδ-dimethyl-D-ornithine dPip D-pipecolic acid, D-homoPro dSer(OBn) O-benzyl-D-serine dTic (3R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid dTiq D-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid dTyr(OAllyl) O-allyl-D-tyrosine dTyr(OBn) O-benzyl-D-tyrosine EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride Fmoc 9-fluorenylmethoxycarbonyl HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HCTU 2-(6-chloro-1H-benzotriazole-1-yl)- 1,1,3,3-tetramethylaminium hexafluorophosphate HFIP 1,1,1,3,3,3-hexafluoro-2-propanol His(Bn) Nτ-benzyl-histidine HomocycloLeu homocyclo leucine, 1-Aminocyclohexanecarboxylic acid Hyp trans-4-hydroxyproline, (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid Hyp(OBn) O-benzyl-trans-4-hydroxyproline MeAsp N-methyl aspartic acid MebetaHomoLys N-methyl β-homoLysine MebetaHomoLys(Me)2 Nα-methyl-Nε-dimethyl-β-homoLysine MeLeu N-methyl leucine MeMet N-methyl methionine MePhe N-methyl phenylalanine metaY(Opr) metaTyrosine MeThr N-methyl threonine MeTyr N-methyl tyrosine NMP N-methylpyrrolidone Nosyl chloride 2-nitrobenzenesulfonyl chloride Nva norvaline Orn(acetamide) Nδ-acetamide-ornithine Orn(benzamide) Nδ-benzamide-ornithine Orn(ethylcarbamate) Nδ-ethylcarbamate-ornithine Orn(methanesulfonamide) Nδ-methanesulfonamide-ornithine Orn(pentyl amide) Nδ-pentyl amide-ornithine PDA 1,4-phenyldiacetic acid Pen penicillamine, β,β-dimethyl-cysteine Pip pipecolic acid, homoPro Sar sarcosine, N-methyl glycine tertbutylAla β-tert-butyl alanine, neopentylglycine TFA trifluoroacetic acid TFE 2,2,2-Trifluoroethanol THF tetrahydrofuran Thr(OBn) O-benzyl-threonine Thr(OEt) O-ethyl-threonine Thr(OMe) O-methyl-threonine Tic (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid TIS triisopropylsilane Tyr(2-methoxy diaryl ether) O-2-methoxy-phenyl-tyrosine Tyr(2-tolyl diaryl ether) O-2-methyl-phenyl-tyrosine Tyr(3,4-difluoro diaryl ether) O-3,4-difluoro-phenyl-tyrosine Tyr(3,4-dimethyl diaryl ether) O-3,4-dimethyl-phenyl-tyrosine Tyr(3-CO2Me diaryl ether) O-3-methylester-phenyl-tyrosine Tyr(3-fluoro diaryl ether) O-3-fluoro-phenyl-tyrosine Tyr(3-methoxy diaryl ether) O-3-methoxy-phenyl-tyrosine Tyr(3-methyl diaryl ether) O-3-methyl-phenyl-tyrosine Tyr(4-CF3 diaryl ether) O-4-trifluoromethyl-phenyl-tyrosine Tyr(4-CO2H diaryl ether) O-4-carboxylate-phenyl-tyrosine Tyr(4-CO2Me diaryl ether) O-4-methylester-phenyl-tyrosine Tyr(4-fluoro diaryl ether) O-4-fluoro-phenyl-tyrosine Tyr(4-methoxy diaryl ether) O-4-methoxy-phenyl-tyrosine Tyr(OAllyl) O-allyl-tyrosine Tyr(OPh) O-phenyl-tyrosine vinyl-Br-Leu 2-amino-4-bromo-4-pentenoic acid

The term “suitable substituent” as used in the context of the present invention is meant to include independently H; hydroxyl; cyano; alkyl, such as lower alkyl, such as methyl, ethyl, propyl, n-butyl, t-butyl, hexyl and the like; alkoxy, such as lower alkoxy such as methoxy, ethoxy, and the like; aryloxy, such as phenoxy and the like; vinyl; alkenyl, such as hexenyl and the like; alkynyl; formyl; haloalkyl, such as lower haloalkyl which includes CF₃, CCl₃ and the like; halide; aryl, such as phenyl and napthyl; heteroaryl, such as thienyl and furanyl and the like; amide such as C(O)NR_(a)R_(b), where R_(a) and R_(b) are independently selected from lower alkyl, aryl or benzyl, and the like; acyl, such as C(O)—C₆H₅, and the like; ester such as —C(O)OCH₃ the like; ethers and thioethers, such as O-Bn and the like; thioalkoxy; phosphino; and —NR_(a)R_(b), where R_(a) and R_(b) are independently selected from lower alkyl, aryl or benzyl, and the like. It is to be understood that a suitable substituent as used in the context of the present invention is meant to denote a substituent that does not interfere with the formation of the desired product by the processes of the present invention.

As used in the context of the present invention, the term “lower alkyl” as used herein either alone or in combination with another substituent means acyclic, straight or branched chain alkyl substituent containing from one to six carbons and includes for example, methyl, ethyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, and the like. A similar use of the term is to be understood for “lower alkoxy”, “lower thioalkyl”, “lower alkenyl” and the like in respect of the number of carbon atoms. For example, “lower alkoxy” as used herein includes methoxy, ethoxy, t-butoxy.

The term “alkyl” encompasses lower alkyl, and also includes alkyl groups having more than six carbon atoms, such as, for example, acyclic, straight or branched chain alkyl substituents having seven to ten carbon atoms.

The term “aryl” as used herein, either alone or in combination with another substituent, means an aromatic monocyclic system or an aromatic polycyclic system. For example, the term “aryl” includes a phenyl or a napthyl ring, and may also include larger aromatic polycyclic systems, such as fluorescent (eg. anthracene) or radioactive labels and their derivatives.

The term “heteroaryl” as used herein, either alone or in combination with another substituent means a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur and which form an aromatic system. The term “heteroaryl” also includes a polycyclic aromatic system comprising a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur.

The term “cycloalkyl” as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent that includes for example, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The term “cycloalkyl-alkyl-” as used herein means an alkyl radical to which a cycloalkyl radical is directly linked; and includes, but is not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 1-cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1-cyclohexylethyl and 2-cyclohexylethyl. A similar use of the “alkyl” or “lower alkyl” terms is to be understood for aryl-alkyl-, aryl-loweralkyl- (eg. benzyl), -lower alkyl-alkenyl (eg. allyl), heteroaryl-alkyl-, and the like as used herein. For example, the term “aryl-alkyl-” means an alkyl radical, to which an aryl is bonded. Examples of aryl-alkyl- include, but are not limited to, benzyl (phenylmethyl), 1-phenylethyl, 2-phenylethyl and phenylpropyl.

As used herein, the term “heterocycle”, either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a three- to seven-membered saturated or unsaturated (including aromatic) cyclic compound containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Examples of such heterocycles include, but are not limited to, aziridine, epoxide, azetidine, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, hydantoin, diazepine, imidazole, isoxazole, thiazole, tetrazole, piperidine, piperazine, homopiperidine, homopiperazine, 1,4-dioxane, 4-morpholine, 4-thiomorpholine, pyridine, pyridine-N-oxide or pyrimidine, and the like.

The term “alkenyl”, as used herein, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a double bond.

Examples of such radicals include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl.

The term “alkynyl”, as used herein is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a triple bond. Examples of such radicals include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl.

The term “alkoxy” as used herein, either alone or in combination with another radical, means the radical —O—(C_(1-n))alkyl wherein alkyl is as defined above containing 1 or more carbon atoms, and includes for example methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. Where n is 1 to 6, the term “lower alkoxy” applies, as noted above, whereas the term “alkoxy” encompasses “lower alkoxy” as well as alkoxy groups where n is greater than 6 (for example, n=7 to 10). The term “aryloxy” as used herein alone or in combination with another radical means —O-aryl, wherein aryl is defined as noted above.

A protecting group or protective group is a substituent introduced into a molecule to obtain chemoselectivity in a subsequent chemical reaction. Many protecting groups are known in the art and a skilled person would understand the kinds of protecting groups that would be incorporated and could be used in connection with the methods described herein. In “protecting group based peptide synthesis”, typically solid phase peptide synthesis, the desired peptide is prepared by the step-wise addition of amino acid moieties to a building peptide chain. The two most widely used protocols, in solid-phase synthesis, employ tert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) as amino protecting groups. Amino protecting groups generally protect an amino group against undesirable reactions during synthetic procedures and which can later be removed to reveal the amine. Commonly used amino protecting groups are disclosed in Greene, T. W. et al., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons (1999). Amino protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; alkoxy- or aryloxy-carbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, .alpha.-,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and sibyl groups such as trimethylsilyl and the like. Amine protecting groups also include cyclic amino protecting groups such as phthaloyl and dithiosuccinimidyl, which incorporate the amino nitrogen into a heterocycle. Typically, amino protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, Alloc, Teoc, benzyl, Fmoc, Boc and Cbz. It is well within the skill of the ordinary artisan to select and use the appropriate amino protecting group for the synthetic task at hand.

In some embodiments, R¹ is H.

In some embodiments, R² or R³ is covalently linked to R¹ to form proline having NR¹ as the N-terminus.

In some embodiments, R² and R³ are not both H.

In some embodiments, R² and R³ are each independently selected from the group consisting of amino acid chains of a proteinogenic or a non-proteinogenic alpha-amino acids.

In some embodiments, R² and R³ are H and CH₃ respectively or vice versa.

In some embodiments, R² or R³ is —CH2-S—R^(s), wherein R^(s) is selected from lower alkyl; lower amino alkyl; aryl; heteroaryl; alkenyl; or heterocycle; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents;

preferably R^(s) is phenyl or phenyl substituted with lower alkyl, halogen; or lower amino alkyl.

In some embodiments, R⁴ and R⁵ are not both H.

In some embodiments, R** and R*** are not both H.

In some embodiments, R⁴ and R⁵ are each independently H, or C(O)—NHR^(t), wherein R^(t) is H or a lower alkyl. Preferably, R^(t) is tert-butyl or H.

In some embodiments, R⁶ is H.

In some embodiments, R⁶ and either R⁸ or R⁹ form a ring resulting in a proline residue having N—R⁶ as its N-terminus.

In some embodiments, n is 1.

In some embodiments, Z along with L and —C═O is any one of SEQ ID NOs. 1-380.

In some embodiments, X¹ is Leu.

In some embodiments, X² is Asp.

In some embodiments, X³ is Thr.

In some embodiments, X³ is Val.

In some embodiments, X³ is Ile.

In some embodiments, X^(y) and X^(z) are each independently a proteinogenic or non-proteinogenic alpha-amino acid.

In some embodiments, X^(z) is a proteinogenic or non-proteinogenic beta-amino acid.

In some embodiments, X^(z) is betaHomoLys or MethylbetaHomoLys.

In some embodiments, X^(y) and X^(z) are each a primary amino acid.

In some embodiments, X^(y) and X^(z) are each any amino acid listed under column X^(y) and column X^(z) respectively of Table 1B.

In various embodiments, the compound is any one of compounds 1-397.

In certain embodiments, there is provided pharmaceutically acceptable salts of the compounds described herein. The term “pharmaceutically acceptable salt,” as used herein, represents salts or zwitterionic forms of the compounds of the present invention which are water or oil-soluble or dispersible, which are suitable for treatment of diseases without undue toxicity, irritation, and allergic response; which are commensurate with a reasonable benefit/risk ratio, and which are effective for their intended use. The salts can be prepared during the final isolation and purification of the compounds or separately by treatment of an amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Also, amino groups in the compounds of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. In certain embodiments, any of the peptide compounds described herein are salt forms, e.g., acetate salts.

In an aspect, there is provided, a pharmaceutical composition comprising a compound described herein along with the pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for any one of oral delivery, topical delivery and parenteral delivery.

As used herein, “pharmaceutically acceptable carrier” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.

In an aspect, there is provided, a method of treating inflammation or an autoimmune disease in a patient, comprising administering to the patient a therapeutically effective amount of the compound described herein. Preferably the inflammation or an autoimmune disease is gastrointestinal.

In an aspect, there is provided, a method for treating a condition in a patient associated with a biological function of an α4β7 integrin, the method comprising administering to the patient a therapeutically effective amount of the compound described herein.

In some embodiments, the condition or disease is Inflammatory Bowel Disease (IBD), ulcerative colitis, Crohn's disease, Celiac disease (nontropical Sprue), enteropathy associated with seronegative arthropathies, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, radiotherapy, chemotherapy, pouchitis resulting after proctocolectomy and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, primary sclerosing cholangitis, human immunodeficiency virus (HIV) infection in the GI tract, eosinophilic asthma, eosinophilic esophagitis, gastritis, colitis, microscopic colitis, graft versus host disease, colitis associated with radio- or chemo-therapy, colitis associated with disorders of innate immunity as in leukocyte adhesion deficiency-1, chronic granulomatous disease, glycogen storage disease type 1b, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, and Wiskott-Aldrich Syndrome, or pouchitis resulting after proctocolectomy and ileoanal anastomosis and various forms of gastrointestinal cancer, osteoporosis, arthritis, multiple sclerosis, chronic pain, weight gain, and depression. In another embodiment, the condition is pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma or graft versus host disease.

In preferable embodiments, is an inflammatory bowel disease, such as ulcerative colitis or Crohn's disease.

In an aspect, there is provided, a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the compound described herein, wherein the disease or condition is a local or systemic infection of a virus or retrovirus.

In some embodiments, the a virus or retrovirus is echovirus 1 and 8, echovirus 9/Barty Strain, human papilloma viruses, hantaviruses, rotaviruses, adenoviruses, foot and mouth disease virus, coxsackievirus A9, human parechovirus 1 or human immunodeficiency virus type 1.

In an aspect, there is provided, a method for treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of the compound described herein, wherein the hepatitis A, B or C, hepatic encephalopathy, non-alcoholic steatohepatitis, cirrhosis, variceal bleeding, hemochromatosis, Wilson disease, tyrosinemia, alpha-1-antitrypsin deficiency, glycogen storage disease, hepatocellular carcinoma, liver cancer, primary biliary cholangitis, primary sclerosing cholangitis, primary biliary sclerosis, biliary tract disease, autoimmune hepatitis, or graft-versus-host disease.

In some embodiments, the compound inhibits binding of α4β7 integrin to MAdCAM. Preferably, the compound selectively inhibits binding of α4β7 integrin to MAdCAM.

In any embodiment, the patient is preferably a human.

As used herein, the terms “disease”, “disorder”, and “condition” may be used interchangeably.

As used herein, “inhibition,” “treatment,” “treating,” and “ameliorating” are used interchangeably and refer to, e.g., stasis of symptoms, prolongation of survival, partial or full amelioration of symptoms, and partial or full eradication of a condition, disease or disorder in a subject, e.g., a mammal.

As used herein, “prevent” or “prevention” includes (i) preventing or inhibiting the disease, injury, or condition from occurring in a subject, e.g., a mammal, in particular, when such subject is predisposed to the condition but has not yet been diagnosed as having it; or (ii) reducing the likelihood that the disease, injury, or condition will occur in the subject.

As used herein, “therapeutically effective amount” refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.

In some embodiments, the compound is administered by a form of administration selected from the group consisting of oral, intravenous, peritoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, nebulization, sublingual, buccal, parenteral, rectal, vaginal, and topical.

In some embodiments, the compound is administered as an initial does followed by one or more subsequent doses and the minimum interval between any two doses is a period of less than 1 day, and wherein each of the doses comprises an effective amount of the compound.

In some embodiments, the effective amount of the compound is the amount sufficient to achieve at least one of the following selected from the group consisting of: a) about 50% or greater saturation of MAdCAM binding sites on α4β7 integrin molecules; b) about 50% or greater inhibition of α4β7 integrin expression on the cell surface; and c) about 50% or greater saturation of MAdCAM binding sites on α4β7 molecules and about 50% or greater inhibition of α4β7 integrin expression on the cell surface, wherein i) the saturation is maintained for a period consistent with a dosing frequency of no more than twice daily; ii) the inhibition is maintained for a period consistent with a dosing frequency of no more than twice daily; or iii) the saturation and the inhibition are each maintained for a period consistent with a dosing frequency of no more than twice daily.

In some embodiments, the compound is administered at an interval selected from the group consisting of around the clock, hourly, every four hours, once daily, twice daily, three times daily, four times daily, every other day, weekly, bi-weekly, and monthly.

The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.

EXAMPLES

Methods and Materials

Synthesis

Methods applicable for making the cyclic peptides described herein can be found generally in Applicant's PCT Publication No. WO 2010/105363 and in an application filed on the same day herewith titled “Fragment Synthesis of Cyclic Peptides” (U.S. patent application Ser. No. 15/775,319) and claiming common priority to U.S. Provisional Application No. 62/254003 filed on Nov. 11, 2015.

More specifically, the below protocols were used to synthesize each of the compounds as indicated in Table S1.

Protocol A: General Nacellin Synthesis

1. Preparation of resin: Fmoc amino acid (1.1 eq. with respect to resin) was dissolved in CH₂Cl₂ (10 mL/g of resin). If the amino acid did not dissolve completely, DMF was added slowly dropwise until a homogeneous mixture persisted upon stirring/sonication. The 2-chlorotrityl resin was allowed to swell in CH₂Cl₂ (5 mL/g of resin) for 15 minutes. The CH₂Cl₂ was then drained and the Fmoc amino acid solution was added to the vessel containing the 2-Cl Trt resin. DIPEA was added (2 eq. with respect to the amino acid) and the vessel was agitated for five minutes. Another 2 eq. of DIPEA was then added and the vessel was left to agitate for an additional 60 minutes. The resin was then treated with methanol (1 mL/g of resin) to endcap any remaining reactive 2-Cl Trt groups. The solution was mixed for 15 minutes, drained and then rinsed with CH₂Cl₂ (x3), DMF (x3), CH₂Cl₂ (x2), and MeOH (x3). The resin was then dried under vacuum and weighed to determine the estimated loading of Fmoc amino acid.

2. Preparation of linear peptide sequence via manual or automated synthesis: Fully protected resin-bound peptides were synthesized via standard Fmoc solid-phase peptide chemistry manually or using an automated peptide synthesizer. All N-Fmoc amino acids were employed.

a. Fmoc deprotection: the resin was treated with 20% piperidine in NMP twice, for 5 and 10 minutes respectively, with consecutive DMF and NMP washes after each addition.

b. Fmoc amino acid coupling: the resin was treated with 3 eq. of Fmoc amino acid, 3 eq. of HATU and 6 eq. of DIPEA in NMP for 60 minutes. For difficult couplings, a second treatment with 3 eq. of Fmoc amino acid, 3 eq. of HATU and 6 eq. of DIPEA in NMP for 40 minutes was employed.

3. General cleavage with retention of protecting groups: Once the desired linear sequence was synthesized, the resin was treated with either 1.) 1:3, HFIP:CH₂Cl₂ or 2.) 5% TFA in CH₂Cl₂, twice for 30 minutes each, to afford cleavage from the solid support. The solvent was then removed, followed by trituration twice with chilled tent-butyl methyl ether (or diethyl ether/hexanes) to give the desired product. The purity was then analyzed by reverse-phase LCMS.

Protocol B: Preparation of N-Alkylated Fmoc Amino Acid Building Blocks

1. Resin prep: see protocol A, step 1

2. Fmoc deprotection: see protocol A, step 2a

3. Nosyl protection: The deprotected resin was stirred in CH₂Cl₂ (5 mL/mmol of resin) and DIPEA (6.5 eq.). A solution of Nosyl chloride (4.0 eq.) was added slowly, dropwise, over 30 minutes, to avoid a rapid exothermic reaction. After the addition was complete, stirring was continued at room temperature for three hours. The resulting nosyl-protected resin was filtered and washed with CH₂Cl₂, MeOH, CH₂Cl₂, and THF.

4. N-Methylation: To a suspension of resin in THF (10 mL/mmol of resin) was added a solution of triphenylphosphine (5 eq.) in THF (2 M) and MeOH (10 eq.). The stirring suspension was cooled in an ice bath. A solution of DIAD (5 eq.) in THF (1 M) was added dropwise, via addition funnel. After addition was complete the bath was removed and the reaction was stirred at room temperature for an additional 90 minutes. The resin was filtered, washed with THF (x4), CH₂Cl₂ (x3), and THF (x2).

5. Nosyl-deprotection: To a suspension of resin in NMP (10 mL/mmol of resin) was added 2-mercaptoethanol (10.1 eq.) and DBU (5.0 eq.). The solution became a dark green colour. After five minutes, the resin was filtered, washed with DMF until washes ran colourless. This procedure was repeated a second time, and the resin was then washed a final time with CH₂Cl₂.

6. Fmoc protection: To a suspension of resin in CH₂Cl₂ (7 mL/mmol of resin) was added a solution of Fmoc-Cl (4 eq.) in CH₂Cl₂ (7 mL), and DIPEA (6.1 eq.). The suspension was stirred at room temperature for four hours then filtered and washed with CH₂Cl₂ (x2), MeOH (x2), CH₂Cl₂ (x2), and Et₂O (x2).

7. Cleavage from resin: see protocol A, step 3

Protocol C: Reductive Amination

1. Fmoc Weinreb amide formation: a mixture of Fmoc amino acid (1 mmol), N,O-dimethylhydroxylamine.HCl (1.2 eq.), and HCTU (1.2 eq.) in CH₂Cl₂ (6.5 mL), was cooled to 0° C. DIPEA (3 eq.) was then slowly added to the stirring mixture. The cooling bath was removed and the reaction was stirred at room temperature for 16 h. A 10% solution of HCl (4 mL) was added resulting in the formation of a precipitate, which was removed through filtration. The filtrate was washed with 10% HCl (3×4 mL) and brine (2×4 mL). The organic phase was then dried over Na₂SO₄. The solvent was removed under reduced pressure to give crude Fmoc Weinreb amide, which was used in the next reaction without purification.

2. Fmoc amino aldehyde formation: lithium aluminum hydride powder (3 eq.) was placed in a dry flask. THF (Sigma-Aldrich, 250 ppm of BHT, ACS reagent>99.0%, 6.5 mL) was added, and the resulting slurry was cooled to −78° C., with stirring. To the slurry was added a solution of the Fmoc Weinreb amide in THF (10 mL). The reaction vessel was transferred to an ice/water bath, and maintained at 0° C. for 1 h. To the reaction at 0° C., was added dropwise acetone (1.5 mL), then H₂O (0.25 mL) and then the reaction was left to stir for an additional hour at room temperature. The mixture was filtered through Celite, washed with EtOAc (10 mL) and MeOH (10 mL), and the filtrate was concentrated. The crude material was dissolved in CHCl₃ (6.5 mL) and washed with brine (2×3 mL) and the organic phase was then dried over Na₂SO₄, filtered and concentrated to give the Fmoc amino aldehyde.

3. Reductive amination on-resin: the linear peptide on-resin was placed in a solid-phase peptide synthesis reaction vessel and diluted with DMF (22 mL/g of resin). The Fmoc aldehyde (4.0 eq.) was added and the reaction was left to shake overnight. The solution was then drained and the resin was washed with CH₂Cl₂ (x3) and DMF (x3). The resin was then diluted with a mixture of MeOH/CH₂Cl₂ (22 mL/g of resin, 1:3 ratio) and NaBH₄(7 eq.) was subsequently added. The mixture was left to shake for four hours, then the solution was drained and the resin was washed with CH₂Cl₂ (x3) and DMF (x3).

Protocol D: Fragment-Based Macrocyclization

In a two-dram vial, 0.1 mmol of the linear peptide and DEPBT (1.5 eq.) were dissolved in 5 mL of freshly distilled THF (0.02 M). DIPEA (3 eq.) was then added and the reaction mixture was left to stir overnight at room temperature (16 h). Tetraalkylammonium carbonate resin (6 eq.) was then added to the reaction mixture and stirring was continued for an additional 24 h. The reaction was then filtered through a solid-phase extraction vessel and rinsed with CH₂Cl₂ (2 mL). The filtrate and washes were combined and the solvent was removed under reduced pressure.

Protocol E: Aziridine Aldehyde-Based Macrocyclization

The linear peptide was dissolved in TFE (if solubility problems were encountered, a 50:50 mixture of TFE:CH₂Cl₂ was used for the cyclization). Then 0.6 eq. of (S)-aziridine-2-carboxaldehyde dimer (prepared as per literature protocol: J. Am. Chem. Soc. 2006, 128 (46), 14772-14773 and Nat. Protoc. 2010, 5 (11), 1813-1822) as a TFE stock solution (0.2 M) was added, giving a final reaction mixture concentration of 0.1 M. tert-Butyl isocyanide (1.2 eq.) was then added and the reaction mixture was stirred for four hours. Progress was analyzed along the way via LC-MS.

Protocol F: Nucleophilic Ring-Opening of Acyl Aziridine, Post Macrocyclization

a.) Thioacetic acid/thiobenzoic acid: thio acid (4 eq.) was added to the crude reaction mixture. Reaction progress was monitored by LC-MS, and was generally complete after 1-2 hours.

Or alternatively, b.) Thiophenol: thiophenol (4 eq.) and DIPEA (4 eq.) were added to the crude cyclization mixture. Reaction progress was monitored by LC-MS, and was generally complete after 1-2 hours. Solvent was removed under reduced pressure and dried under vacuum. Crude material was either triturated with Et₂O/hexanes or TBME, or alternatively, diluted with H₂O, frozen and lyophilized.

Protocol G: General Suzuki Coupling, Post Macrocyclization

An iodo-Phe-containing macrocycle (0.1 mmol), Na₂CO₃ (2 eq.), substituted boronic acid (1.1 eq.) and 4 mL of water:acetonitrile (1:1 ratio) were combined in a microwave vial. The mixture was degassed via N₂ flow for 10 minutes. While under N₂, silicon based Pd-catalyst (Siliacat-DPP Pd heterogenous catalyst, 0.05 eq.) was added. The reaction vial was sealed and placed in the microwave for 10 minutes at 150° C. Reaction progress was monitored by LCMS. Once complete, the reaction was filtered through a Celite plug and the solvent was removed under reduced pressure.

Protocol H: General Ulmann Coupling, Post Macrocyclization

Under inert atmosphere, the peptide macrocycle (0.018 mmol) was placed in a 2-dram vial containing 2 mL of dry CH₂Cl₂. Cu(OAc)₂ (1 eq.), benzene boronic acid (2 eq.) and 4 Å (oven-dried) molecular sieves were then added to the vial followed by DIPEA (4 eq.). The contents of the vial were stirred at room temperature overnight. The reaction progress was assessed by LCMS. Once the reaction was deemed complete, the mixture was filtered through a Celite plug and the solvent was removed under reduced pressure.

Protocol I: General Global Deprotection and Cleavage

Deprotection of the side chain protecting groups was achieved by dissolving the peptides in 2 mL of a cleavage cocktail consisting of TFA:H₂O:TIS (95:2.5:2.5) for two hours. Subsequently, the cleavage mixture was evaporated under reduced pressure and the peptides were precipitated twice from chilled diethyl ether/hexanes (or tert-butyl methyl ether).

Protocol J: General Cleavage of Reductively-Labile Protecting Groups

a.) Pd/C and formic acid debenzylation: the benzyl protected macrocycle (0.35 mmol) was dissolved in MeOH (8 mL) with 10% formic acid, 50% wt. Pd/C (1 mg) and heated to 55° C. Once the reaction was deemed complete, the mixture was filtered through a Celite plug, washed with MeOH and the solvent was removed under reduced pressure.

Or alternatively, b.) Raney Ni desulfurization/debenzylation: Raney Ni slurry (1-2 mL) was added directly to the cyclization reaction mixture and stirred vigorously overnight. The vial was then centrifuged and the liquid was transferred using a pipette to a tared vial. MeOH was added to the vial containing Raney Ni. The vial was then sonicated, vortexed, and centrifuged. Again, the liquid was transferred to a tared vial. This process was repeated with EtOAc and then a final time with MeOH. The combined washes were then removed under reduced pressure and the residue dried under vacuum.

Protocol K: Amidation of Side Chain, Post Macrocyclization

Macrocycle (0.021 mmol) was dissolved in 1 mL of CH₃CN. K₂CO₃ (5 eq.) and acid chloride (2 eq.) were then added and the reaction mixture was left to stir at room temperature overnight. Reaction progress was checked by LC-MS in the morning. Upon completion, the solvent was removed by reduced pressure.

Protocol L: Fluorescent Dye Attachment

The macrocycle (4 μmol) was dissolved in DMSO (200 μL). DIPEA (5 eq.) was then added. In a separate vial, 5 mg of fluorescent dye as the NHS ester was dissolved in 200 μL of DMSO. The macrocycle solution was then added to the solution of the fluorescent label. The reaction mixture was stirred overnight. Reaction progress was checked by LC-MS in the morning and then the solvent was removed by lyophilization.

Protocol M: Purification Methods

All macrocycles were purified using reverse-phase flash column chromatography using a 30 g RediSep C18 Gold Column. The gradient consisted of eluents A (0.1% formic acid in double distilled water) and B (0.1% formic acid in HPLC-grade acetonitrile) at a flow rate of 35 mL/min.

Integrin α4β7—MAdCAM-1 ELISA Competition Assay

Definitions and Acronyms

BSA: Bovine serum albumin

DMSO: Dimethyl sulfoxide

HRP: Horseradish peroxydase

PBS: Phosphate buffered saline

TMB: 3,3′,5,5′-tetramethylbenzidine

Required Products

Product Company Catalog # Recombinant human integrin α4β7 R&D Systems 5397-A3-050 Recombinant human MAdCAM-1 Fc R&D Systems 6056-MC-050 Chimera Goat anti-human IgG Fc specific (HRP) Abcam Ab97225 NaHCO₃ BDH ACS 804 Na₂CO₃ BDH ACS 777 Tris-Cl Fisher BP 153-1 NaCl EMD 7710 MnCl₂•4 H₂O Sigma M-3634 BSA Omni Pur 2930 H₂SO₄ Fisher A300-212 KCl BDH ACS 645 Na₂HPO₄ Sigma S-0876 KH₂PO₄ Sigma P3786 DMSO SAFC RES2166D- A101X TMB (SureBlue Reserve) KPL 53-00-00 Tween 20 Sigma D7949 96-well flat bottomed plates Microlon Greiner 655001 200 (med binding) 96-well round bottomed plates Costar 3797

Specific Material

Equipment Feature Plate washer HydroSpeed Tecan Plate reader Infinite 1000 Tecan Humidified chamber Plate shaker 85 rpm

Solutions

Carbonate buffer pH 9,6 (50 mM), Tris-Cl (1 M), Blocking buffer (50 mM Tris, 150 mM NaCl, 1 mM MnCl₂, 1% BSA, 0.05% Tween), Assay buffer (50 mM Tris, 150 mM NaCl, 1 mM MnCl₂, 0.1% BSA, 0.05% Tween), Wash buffer (50 mM Tris, 100 mM NaCl, 1 mM MnCl₂, 0.05% Tween), H₂SO₄ 1M, PBS, Dilution solution (400 μL DMSO in 40 mL of Assay buffer)

Protocol

-   -   1.1 Preparation of α4β7.         -   1.1.1 Add 500 μL of PBS to the vial. Do not vortex and leave             it on ice for 15 minutes. Mix by inversion before use. Keep             at 4° C. for up to 1 month.             -   Final concentration is 100 pg/mL     -   1.1 Preparation of MadCam-Fc.         -   1.1.1 Add 500 μL of Assay buffer to the vial. Do not vortex             and leave it on ice for 15 minutes. Mix by inversion before             use. Keep at 4° C. for up to 1 month.             -   Final concentration is 100 μg/mL     -   1.2 Absorption of α4β7 onto the plate.         -   1.2.1 Dilute α4β7 at a concentration of 1 μg/mL in fresh             carbonate buffer.         -   1.2.2 Using a 12-chanel pipette, distribute 100 μL per well.         -   1.2.3 Incubate the plate 16-18 hours at 4° C. in a             humidified chamber.     -   1.3 Blocking step         -   1.3.1 Pour the content of the plate over the sink followed             by gently dabbing the plate upside down on a blotting paper.         -   1.3.2 Add 250 μL of blocking buffer per well.         -   1.3.3 Incubate at 23-25° C. for 1 hour.         -   1.3.4 Wash the plate using the plate washer: method “MadCam”     -   1.4 Preparation of Compound Dilutions         -   1.4.1 During the blocking step, prepare serial dilutions of             the test compounds     -   Add 100 μL of dilution solution in wells of rows #1 to #11 in a         dilution plate     -   Add 198 μL of assay buffer in wells of row #12     -   Add 2 μL of test compounds in the appropriate well in row #12     -   Using a multichannel pipette, mix thoroughly the content of the         wells in row #12     -   Do serial dilutions, starting with row #12 to #1 by transferring         100 μL well to well. Mix thoroughly between each transfer.         -   1.4.2 Immediately after the wash step (7.3.4), transfer 50             μL of the compounds from the dilution plate to the test             plate.         -   1.4.3 Add 100 μL of assay buffer in well A1 and A2 (Blank).         -   1.4.4 Add 50 μL of dilution solution in well A3 to A12             (Maximal binding).     -   1.5 Addition of MadCam-Fc         -   1.5.1 Dilute the MadCam-Fc at 1 μg/mL in Assay buffer         -   1.5.2 Add 50 μL to each well of the test plate (except wells             A1 and A2)         -   1.5.3 Incubate at 23-25° C. for 2 hours under agitation (85             rpm).         -   1.5.4 Wash the plate using the plate washer: method “MadCam”     -   1.6 Addition of anti-human IgG (Fc specific)-HRP         -   1.6.1 Dilute the anti-human IgG Fc specific-HRP (1:2000) in             Assay buffer         -   1.6.2 Add 100 pL of diluted anti-human IgG in each well.         -   1.6.3 Incubate at 23-25° C. for 1 hour under agitation (85             rpm).         -   1.6.4 Wash the plate using the plate washer: method “MadCam”     -   1.7 Revelation         -   1.7.1 Add 100 μL of TMB.         -   1.7.2 Stop the reaction after 2 minute-incubation by adding             50 μL H₂SO₄ 1M         -   1.7.3 Read the OD_(450 nm) with the plate reader.

Integrin α4β1—VCAM-1 ELISA Competition Assay

Definitions and Acronyms

BSA: Bovine serum albumin

DMSO: Dimethyl sulfoxide

HRP: Horseradish peroxydase

PBS: Phosphate buffered saline

TMB: 3,3′,5,5′-tetramethylbenzidine

Required Products

Product Company Catalog # Recombinant human integrin α4β1 R&D Systems 5668-A4-050 Recombinant human VCAM-1 Fc R&D Systems 862-VC-100 Chimera Goat anti-human IgG Fc specific (HRP) Abcam Ab97225 NaHCO₃ BDH ACS 804 Na₂CO₃ BDH ACS 777 Tris-Cl Fisher BP 153-1 NaCl EMD 7710 MnCl₂•4 H₂O Sigma M-3634 BSA Omni Pur 2930 H₂SO₄ Fisher A300-212 KCl BDH ACS 645 Na₂HPO₄ Sigma S-0876 KH₂PO₄ Sigma P3786 DMSO SAFC RES2166D- A101X TMB (Slow) Thermo Fisher 34024 Tween 20 Sigma D7949 96-well flat bottomed plates Microlon Greiner 655001 200 (med binding) 96-well round bottomed plates Costar 3797

Specific Material

Equipment Feature Plate washer HydroSpeed Tecan Plate reader Infinite 1000 Tecan Humidified chamber Plate shaker 85 rpm

Solutions

Carbonate buffer pH 9.6 (50 mM), Tris-Cl (1 M), Blocking buffer (50 mM Tris, 150 mM NaCl, 1 mM MnCl₂, 1% BSA, 0.05% Tween), Assay buffer (50 mM Tris, 150 mM NaCl, 1 mM MnCl₂, 0.1% BSA, 0.05% Tween), Wash buffer (50 mM Tris, 100 mM NaCl, 1 mM MnCl₂, 0.05% Tween), H₂SO₄ 1M, PBS, Dilution solution (400 μL DMSO in 40 mL of Assay buffer)

Protocol

-   -   1.8 Preparation of α4β1.     -   1.8.1 Add 500 μL of PBS to the vial. Do not vortex and leave it         on ice for 15 minutes. Mix by inversion before use. Keep at         4° C. for up to 1 month.         -   Final concentration is 100 μg/mL     -   1.9 Preparation of VCam-Fc.         -   1.9.1 Add 500 μL of Assay buffer to the vial. Do not vortex             and leave it on ice for 15 minutes. Mix by inversion before             use. Keep at 4° C. for up to 1 month.             -   Final concentration is 200 μg/mL     -   1.10 Absorption of α4β1 onto the plate.         -   1.10.1 Dilute α4β1 at a concentration of 0.5 μg/mL in fresh             carbonate buffer.         -   1.10.2 Using a 12-chanel pipette, distribute 100 μL per             well.         -   1.10.3 Incubate the plate 16-18 hours at 4° C. in a             humidified chamber.     -   1.11 Blocking step         -   1.11.1 Pour the content of the plate over the sink followed             by gently dabbing the plate upside down on a blotting paper.         -   1.11.2 Add 250 μL of blocking buffer per well.         -   1.11.3 Incubate at 23-25° C. for 1 hour.         -   1.11.4 Wash the plate using the plate washer: method             “MadCam”     -   1.12 Preparation of compound dilutions         -   1.12.1 During the blocking step, prepare serial dilutions of             the test compounds     -   Add 100 μL of dilution solution in wells of rows #1 to #11 in a         dilution plate     -   Add 198 μL of assay buffer in wells of row #12     -   Add 2 μL of test compounds in the appropriate well in row #12     -   Using a multichannel pipette, mix thoroughly the content of the         wells in row #12     -   Do serial dilutions, starting with row #12 to #1 by transferring         100 μL well to well. Mix thoroughly between each transfer.         -   1.12.2 Immediately after the wash step (7.3.4), transfer 50             μL of the compounds from the dilution plate to the test             plate.         -   1.12.3 Add 100 μL of assay buffer in well A1 and A2 (Blank).         -   1.12.4 Add 50 μL of dilution solution in well A3 to A12             (Maximal binding).     -   1.13 Addition of VCam-Fc         -   1.13.1 Dilute the VCam-Fc at 1 μg/mL in Assay buffer         -   1.13.2 Add 50 μL to each well of the test plate (except             wells A1 and A2)         -   1.13.3 Incubate at 23-25° C. for 2 hours under agitation (85             rpm).         -   1.13.4 Wash the plate using the plate washer: method             “MadCam”     -   1.14 Addition of anti-human IgG (Fc specific)-HRP         -   1.14.1 Dilute the anti-human IgG Fc specific-HRP (1:2000) in             Assay buffer         -   1.14.2 Add 100 μL of diluted anti-human IgG in each well.         -   1.14.3 Incubate at 23-25° C. for 1 hour under agitation (85             rpm).         -   1.14.4 Wash the plate using the plate washer: method             “MadCam”     -   1.15 Revelation         -   1.15.1 Add 100 μL of TMB (slow TMB).         -   1.15.2 Stop the reaction after 30 minute-incubation by             adding 50 μL H₂SO₄ 1M         -   1.15.3 Read the OD_(450 nm) with the plate reader.

RPM18866 Cell Adhesion Competition Assay

Material:

-   -   1) Recombinant human MAdCAM-1 Fc Chimera, R&D Systems,         6056-MC-050     -   2) RPMI 8866 cells (grown in RPMI 1640 media supplemented with         10% FBS and 1% Pen/Step)     -   3) RPMI 1640 media, Wisent, 350-000-CL

Protocol:

-   -   1) Coat Maxisorp plate (Nunc 442404) with 100 ul (0.25 ug/ml in         coating buffer) Madcam overnight at 4° C.     -   2) Wash 2× (300 ul) with wash buffer using an e1200 Biohit         8-channel pipette with aspirate speed set to 4 and dispense         speed set to 1.     -   3) Block with 250 ul blocking buffer for 1 hr at RT with the         same pipette settings as above.     -   4) Collect 10M RPMI 8866 cells/plate in 50 ml tube. Top off with         PBS and spin 5 min at 250 rpm.     -   5) Resuspend cells to 10M/ml in PBS containing 5 uM Calcein.     -   6) Incubate cells at 37° C. in the dark for 30 min.     -   7) Top off cells with PBS and spin.     -   8) Resuspend cells to 2 M/ml in neat RPMI 1640 media.     -   9) Prepare compounds in binding buffer (prepare 100 ul per         replicate).     -   10) Empty blocking buffer and wash plate 1× with 300 ul PBS         (pipette settings as in step 2).     -   11) Transfer 50 ul of compound/control and 50 ul of cells to         each well and incubate in the dark for 45 min.     -   12) Read plate on Biotek Neo using FITC 96 bottom read for         pre-wash readings.     -   13) Add 150 ul PBS (pipette settings as in step 2).     -   14) Invert plate and blot on paper towel.     -   15) Gently add 200 ul PBS (pipette settings as in step 2),         invert plate and blot on paper towel.     -   16) Add 100 ul PBS using e300 Biohit 8-channel pipette with         aspirate speed set to 4 and dispense speed set to 1.

17) Read plate on Biotek Neo using FITC 96 bottom read for pre-wash readings.

Buffers

Coating buffer (50 mM carbonate)

Dissolve 420 mg sodium bicarbonate in 100 ml water (Soln 1). Dissolve 270 mb sodium carbonate in 50 ml water (Soln 2). Add Soln 2 to Soln 1 to a pH of 9.6

Wash buffer

0.05% Tween 20 in PBS

Blocking buffer

1% Nonfat Dry Milk in PBS

Binding buffer

1.5 mM CaCl₂

0.5 mM MnCl₂

50 mM Tris-HCl, pH to 7.5 with HCl

Plasma Protein Binding Determination

An equilibrium dialysis (HTDialysis) method was used employing 50% plasma collected from CD-1 mice or Sprague-Dawley rats and incubated with K2EDTA. Test articles were assessed at 1 microM concentration in three replicates. Incubation time as 5 hours and plasma and buffer standards were assessed using LC/MS/MS. Percentage recovery was calculated as (C_(buffer)+C_(plasma))/[average]C_(initial), [average]C_(initial) being measured in triplicate from the test samples prior to dialysis. Percentage of compound unbound was calculated as C_(buffer)/C_(plasma).

Aqueous Solubility Assay

Aqueous solubility was determined by using 1, 0.5 or 0.2 mM (maximum) of test compound in phosphate buffered saline with pH of 7.4. Solutions were incubated for four hours at room temperature and stirred at 600 rpm and run in triplicate. Centrifugation was performed for 15 minutes at 6000 rpm and solution concentration was determined using HPLC-UV (photodiode array detector acquiring between 220 nm, and 300 nm wavelengths).

Cytochrome P450 Inhibition Assay

Human liver microsomes (at 0.25 mg/ml, except for CYP1A2, where a concentration of 0.5 mg/ml was employed) were used to assess the IC₅₀ (in duplicate; concentration range of 0.25 nM to 15 microM) for test compounds on the activity four isoforms of cytochrome P450 (“CYP”): CYP2D6, CYP3A4, CYP2C9 and CYP1A2. The following substrates were employed: dextromethorphan (15 microM, CYP2D6), testosterone (50 microM, CYP3A4), diclofenac (10 microM, CYP2C9) and phenacetin (100 microM, CYP1A2). Control inhibitors were quinidine (0.03 nM to 1.5 microM, CYP2D6), ketoconazole (0.08 nM to 5 microM, CYP3A4), miconazole (0.25 nM to 15 microM, CYP2C9) and alpha-naphoflavone (0.03 nM to 1.5 microM, CYP1A2). Incubation time was 10-20 minutes and metabolites assessed were dextrorphan (CYP2D6), 6-beta-OH-testosterone (CYP3A4), 4′-OH-diclofenac (CYP2C9) and acetaminophen (CYP1A2). Internal standards were labetalol (CYP2D6), loratidine (CYP3A4), carbamazepine (CYP2C9) and metoprolol (CYP1A2). Analyses were performed using standard LC/MS/MS protocols.

In Vivo T Lymphocyte Trafficking Analyses

Animal care committee. The animal care facility employed is accredited by the Canadian Council on Animal Care (CCAC). This study was approved by a certified Animal Care Committee and complied with CACC standards and regulations governing the use of animals for research.

Animals. Female C57BI/6 mice (Charles River, St-Constant, Qc), weighting 16-19 g at delivery were used for this study. Following arrival in the animal facility, all animals were subjected to a general health evaluation. An acclimation period of 7-14 days was allowed before the beginning of the study.

Housing environment. The animals were housed under standardized environmental conditions. The mice were housed in auto-ventilated cages, 2-3 per cage. Each cage was equipped with a manual water distribution system. A standard certified commercial rodent diet was provided ad libitum. Tap water was provided ad libitum at all times. It is considered that there are no known contaminants in the diet and water that would interfere with the objectives of the study. Each cage was identified for the corresponding group, indicating the treatment and the identity of the animals housed in the cage. Mice from different treatment groups were not mixed.

The animal room was maintained at a controlled temperature of 21.5±1° C. and a relative humidity of 40±10%. A controlled lighting system assured 12 hours light, 12 hours dark per day to the animals. Adequate ventilation of 8-10 air changes per hour was maintained.

Administration of DSS. Dextran sulfate sodium (DSS) was administered to C57B1/6 mice through addition to their drinking water at 2-3%. Mice accessed the DSS-treated water ad libitum over a 5-day period. Body weight and disease activity index (“DAI”) were measured on Day 5 in order to distribute DSS-treated animals in two uniform groups prior to dosing. Specific symptoms associated with colitis were scored based on the severity of each particular symptoms: 1—blood in stool (negative hemoccult, positive hemoccult, blood traces in stool visible, rectal bleeding); 2—stool consistency (normal, soft but still formed, very soft, diarrhea); 3—posture and fur (normal; ruffled fur; ruffled fur combined to slight hunched posture and slight dehydration; ruffled fur combined to hunched posture, dehydration and altered walking; moribund (euthanasia is mandatory before the animal reach this point). The overall DAI score was the sum of the three parameters (maximum score of 9). DAI assessment was performed on Day 5 only (prior to dosing).

Oral dosing of the test article and vehicle. On day 6, the test articles were administered in the morning, as a single slow bolus (over approximately 5 seconds) via oral route, according to the procedure of administration of solution by gavage: the animal was firmly restrained. A bulb-tipped gastric gavage needle of 22G was passed through the side of the mouth and was advanced towards the oesophagus. The test articles and the vehicle were dosed orally at 10 mL/kg. Dosing volume was individually adjusted according to the body weight of each animal to reach the target dose.

Intravenous dosing of the test article and the vehicle. On day 6, the test article, DATK32 antibody, and the vehicle were administered in the morning, as a single slow bolus injection (over approximately 5 seconds) via the tail vein, according to the procedure of administration of solution by intravenous administration: the animal was restrained and its tail was warmed prior to dosing. A needle of 30G was used to inject the test article, or the vehicle, through the median tail vein at a dosing volume of 5 mL/kg. Dosing volume was individually adjusted according to the body weight of each animal to reach the target dose of DATK32 control antibody.

Collection of samples. On Day 6, five hours after test article or vehicle dosing, the animals were euthanized by cardiac puncture under general anesthesia, according to the “Guide to the Care and Use of Experimental Animals” published by the CCAC. Blood was transferred in a Sarstedt tube containing EDTA. Mesenteric and peripheral (inguinal, auxiliary and brachial) lymph nodes were collected and transferred on ice to corresponding tubes containing cold PBS. Nodes were kept on ice until tissue preparation.

Cell population labeling. Blood was withdrawn by cardiac puncture and collected on EDTA-coated tubes. Mensenteric lymph nodes (MLN) and peripheral lymph nodes (PLN) were also collected. Mononuclear cells from the tissues were isolated using density gradient (Lympholyte) and they were stained with fluorescent antibodies. The cells (5×104) were first incubated 15 minutes with BD mouse FeBlock (Fcγ III/II Receptor) followed by a 30-minute incubation with specific antibodies. After washes, cells were fixed using BD Fix Solution.

Specific Antibodies Used:

Antibodies Company Catalog # CD3 FITC BD Biosciences 555274 CD4 APC BD Biosciences 553051 CD11a PE BD Biosciences 553121 CD45 PE BD Biosciences 553081 A4β7 PE eBiosciences 12-5887 CD34PE BD Biosciences 551387

Percentage of different subpopulations of T lymphocytes were then analyzed using FACS-Calibur cytometer.

In Vivo Pharmacokinetic Assessments in Rodents

Oral bioavailability of test compounds was conducted by assessing plasma exposure of following one or two oral doses in mice and, in some cases, comparing said plasma exposure with that following a single intravenous dose of the same compound.

More detail on experimental design follows:

Group Test No. & sex Dosing Dose Concentration Volume ID article ID Route of animals Frequency (mg/kg) (mg/mL) (mg/mL) Sample Collection 1 p.o 18 M once 40 4 10 Terminal blood 2 p.o 18 M twice* 40 4 10 (3 mice/time-point) *Dosing will occur 8 hours apart.

In all cases, formulation of test compound was 30% Labrasol in PBS (v/v) for oral dosing and 25% PEG-400 in PBS (v/v) for intravenous dosing.

Collection of peripheral blood proceeded as follows:

Group ID Blood collection time (h) Volume/animal/time-point 1 &2* 0.0833, 0.5, 1, 2, 3 & 5 ~0.6 mL *For Group 2, sample collection will be conducted following the second dose.

In some cases, collections proceeded up to 24 hours. Also note that in a few studies, liver and colon were also collected from mice concomitantly with peripheral blood and on a terminal (and serial) basis.

Other study details include:

Animals: Male CD-1 mice (20-25 g) from Charles River Labs were acclimatized for a minimum of 5 days prior to dosing. Body weights were recorded on the day of dosing.

Food restriction: Animals dosed p.o. were deprived of food overnight and fed ˜2 h following dosing.

Clinical observations: Animals were observed at the time of dosing and each sample collection. Any abnormalities were documented.

Dosing: The formulation containing the test compound were administered p.o. by gavage with disposable feeding needles.

Sample collection: Terminal blood and tissue samples were collected under O2/CO2 anesthesia by cardiac puncture. The colon samples (a 0.5 cm section, 2.5 cm distal to the cecum) will be excised, rinsed with ice cold PBS, blotted and weighed (as applicable). The livers will be blotted and weighed. Plasma, liver and colon samples will be stored frozen at −80 degrees centigrade until bioanalysis.

Sample processing/storage: All blood samples were transferred into K2EDTA tubes on wet ice and centrifuged within 5 min (3200×g for 5 min at 4° C.) to obtain plasma. Samples were stored frozen at −80° C. until bioanalysis.

Sample retention: Plasma samples were analyzed and any remaining samples were stored frozen at −80° C. until the study is completed. Remaining sample were discarded.

Bioanalytical Method Qualification and Sample Analysis:

Matrix: Mouse Plasma

Instrumentation: AB Sciex API 4000 Q-TRAP MS/MS system equipped with an Agilent LC system with a binary pump, a solvent degasser, a thermostatted column compartment, a CTC autosampler and a divert valve installed between the column and mass spectrometer inlet.

Method Qualification:

The determination of the quantification dynamic range using non-zero calibration standards (STDs) in singlet. The STDs will consist of a blank matrix sample (without IS), a zero sample (with IS), and at least 6 non-zero STDs covering the expected range and including the lower level of quantitation (LLOQ).

Three injections of a system suitability sample (neat solution containing the analytes and IS) bracketing the batch.

Method Acceptance Criteria:

At least 75% of non-zero STDs must be included in the calibration curve with all back-calculated concentrations within ±20% deviation from nominal concentrations (±25% for the lower level of quantification, LLOQ).

The correlation coefficient (r) of the calibration curve must be greater than or equal to 0.99.

The area ratio variation between the pre-and post-run injections of the system suitability samples is within ±25%.

Samples which are >1-fold the highest calibration standard, will be diluted and re-assayed along with a corresponding dilution quality control standard.

Sample Analysis Batch:

Three injections of a system suitability sample bracketing the batch

The STDs in ascending order bracketing the study samples and dosing solutions

-   -   1. The study samples     -   2. The dosing solutions diluted as 3 independent dilutions into         blank matrix (mouse plasma) 8-Day Efficacy Study in DSS Model         (Therapeutic) with ET02451-01 (Compound No. 340) and ET02452-01         (Compound No. 341)

Study Design

Cohort N UC Treatment Dose Volume 1 5 DSS vehicle  0 mg/kg 5 mL/kg (p.o.) 2 5 DSS ET02451-01 40 mg/kg 5 mL/kg (p.o.) 3 5 DSS ET02452-01 40 mg/kg 5 mL/kg (p.o.) 4 5 DSS ET02452-01 65 mg/kg 5 mL/kg (i.p.) 5 5 DSS DATK32 15 mg/kg 6 mL/kg (i.p.)

Description of Tested Compounds

Name: Vehicle Labrasol/PBS

Volume: 8.0 mL

Solution: Labrasol (30%)/PBS (70%)

Storage: 4° C.

Name: ET02451 (Compound No. 340)

Volume: 3.7 mL

Solution: 8 mg/mL in Labrasol (30%)/PBS (70%)

Storage: 4° C.

Name: ET02452 (Compound No. 341)

Volume: 3.45 mL

Solution: 8 mg/mL in Labrasol (30%)/PBS (70%)

Storage: 4° C.

Name: ET02452 (Compound No. 341)

Volume: 3.40 mL

Solution: 13 mg/mL in PEG400 (40%)/PBS (60%)

Storage: 4° C.

Name: DATK32 Antibody (eBiosciences #14-5887-85, lot #4282190)

Volume: 5 mL

Solution: 0.5 mg/mL concentrated to 2.5 mg/mL following concentration step

Storage: 4° C.

Name: Vehicle PEG/PBS

Volume: 8.0 mL

Solution: PEG400 (40%)/PBS (60%)

Storage: 4° C.

Animal care committee. The animal care facility employed is accredited by the Canadian Council on Animal Care (CCAC). This study was approved by a certified Animal Care Committee and complied with CACC standards and regulations governing the use of animals for research.

Animals. Female C57BI/6 mice (Charles River, St-Constant, Qc), weighting 16-19 g at delivery were used for this study. Following arrival in the animal facility, all animals were subjected to a general health evaluation. An acclimation period of 7-14 days was allowed before the beginning of the study.

Housing environment. The animals were housed under standardized environmental conditions. The mice were housed in auto-ventilated cages, 2-3 per cage. Each cage was equipped with a manual water distribution system. A standard certified commercial rodent diet was provided ad libitum. Tap water was provided ad libitum at all times. It is considered that there are no known contaminants in the diet and water that would interfere with the objectives of the study. Each cage was identified for the corresponding group, indicating the treatment and the identity of the animals housed in the cage. Mice from different treatment groups were not mixed.

The animal room was maintained at a controlled temperature of 21.5±1° C. and a relative humidity of 40±10%. A controlled lighting system assured 12 hours light, 12 hours dark per day to the animals. Adequate ventilation of 8-10 air changes per hour was maintained.

Oral dosing of the test article and the vehicle. From Day 5 to Day 8, nacellins and the vehicle were administered as a single slow bolus (over approximately 5 seconds) via oral route (p.o.), according to the procedure of administration of solution by gavage: the animal was firmly restrained. A bulb-tipped gastric gavage needle of 22G was passed through the side of the mouth and was advanced towards the oesophagus. The test articles and the vehicle were dosed orally at 5 mL/kg. Dosing volume was individually adjusted according to the body weight of each animal to reach the target dose of ET02451and ET02452 (40 mg/kg).

Intraperitoneal dosing of the test articles and the vehicle. On day 5, DATK32 antibody was administered in only on Day 5, as a single slow bolus (over approximately 5 seconds) via the i.p. route. ET02452 prepared in PEG400 (40%)/PBS (60%) and the i.p. vehicle (PEG400 (40%)/PBS (60%)) were administered from Day 5 to Day 8. Intraperitoneal administration was performed accordingly to the following procedure: the mouse was restrained manually and held with the head and body tilted downward. The tip of the needle (27 G) was inserted through the skin and just past the abdominal wall. A short pull back of the plunger of the syringe was done prior to administration of the solution to make sure that the syringe was not inserted in any abdominal organ (fluid would be pulled back into the syringe in this case). Dosing volume was individually adjusted according to the body weight of each animal to reach the target dose of DATK32 antibody (15 mg/kg) and of ET02452 (65 mg/kg).

Inflammation Score. Once the mouse was euthanized, the colon was collected and its length was measured. Lesion length was also measured. Colon inflammation was scored based on severity of oedema and ulceration.

Disease Activity Index (DAI) assessment. Body weight and DAI were measured on Day 5 in order to distribute DSS-treated animals in two uniform groups prior dosing. Specific symptoms associated to UC were scored based on the severity of each particular symptoms: 1—blood in stool (negative hemoccult, positive hemoccult, blood traces in stool visible, rectal bleeding); 2—stool consistency (normal, soft but still formed, very soft, diarrhea); 3—posture and fur (normal; ruffled fur; ruffled fur combined to slight hunched posture and slight dehydration; ruffled fur combined to hunched posture, dehydration and altered walking; moribund (euthanasia is mandatory before the animal reach this point). The overall DAI score was the sum of the three parameters (maximum score 9). Body weight measurement and DAI assessment were performed also on Day 8 to evaluate the effect of the treatments.

Collection of samples. On Day 8, five hours after ET02451-01, ET02452-01 and vehicle dosing, the animals were euthanized by cardiac puncture under general anesthesia, according to the “Guide to the Care and Use of Experimental Animals” published by the CCAC. Blood was transferred in a Sarstedt tube containing EDTA. Mesenteric and peripheral (inguinal, auxiliary and brachial) lymph nodes were collected and transferred on ice in corresponding tubes containing cold PBS. Nodes were kept on ice until tissue preparation.

Cell population labeling. Blood was withdrawn by cardiac puncture and collected on EDTA-coated tubes. Mensenteric lymph nodes (MLN) and peripheral lymph nodes (PLN) were also collected. Mononuclear cells from the tissues will be isolated using density gradient (Lympholyte) and they were stained with fluorescent antibodies. The cells (5×104) were first incubated 15 minutes with BD mouse FeBlock (Fcγ III/II Receptor) followed by a 30-minute incubation with specific antibodies. After washes, cells were fixed using BD Fix Solution. Percentage of different subpopulations of T lymphocytes were then analysed using FACSCalibur cytometer. Antibodies employed were the same as listed for the single-dose PD studies above.

Results and Discussion

Compounds were synthesized in accordance with the above-noted methods. A selection of compounds were characterized using NMR (not all data shown). A subset of NMR data is provided for select compounds in FIGS. 14-47.

Plasma Protein Binding Determination

The sequestration of nacellins by plasma proteins was relatively low. In rat plasma, free fraction (% unbound) ranged from an 9.5% to 76.9% (mean of 42.6%), whereas in mouse plasma, free fraction ranged from 15.7% to 79.9% (mean of 47.8%). Plasma protein binding for the small molecule positive control, propranolol, was in the normal range of ˜21% (free fraction) in mouse plasma and ˜15% (free fraction) in rat plasma. The compounds assessed include:

ET01792 (Compound No. 5)

ET00762 (an analog of Compound No. 5, in which the phenylalanine residue has been replaced by a tryptophan residue)

ET01813 (Compound No. 12)

ET01827 (Compound No. 15)

% unbound (C_(buffer)/C_(plasma)) List of Mouse plasma % % Control buffer/Buffer at 0.1 uM Compounds 1 2 3 Average sd RSD recovery Recovery Cdonor/Creciever ET01792 64.8 52.9 67.9 61.9 7.9 13%  130% 108% 80% ET00762 80.6 74.4 75.7 76.9 3.3 4% 150%  99% 79% ET01813 24.7 22.2 18.8 21.9 3.0 14%  113% 128% 94% ET01827 10.0 9.3 9.3 9.5 0.4 4% 103%  70% 86% Propranalol 22.8 20.1 20.1 21.0 1.6 7%  93%

% unbound (C_(buffer)/C_(plasma)) List of Mouse plasma % % Compounds 1 2 3 Average sd RSD recovery ET01792 82.9 77.3 79.5 79.9 2.8 4% 97% ET00762 83.3 76.1 70.2 76.5 6.6 9% 94% ET01813 15.8 20.1 21.7 19.2 3.1 16%  89% ET01827 16.2 17.4 13.6 15.7 1.9 12%  143%  Propranalol 14.6 14.7 14.7 14.7 0.1 0% 79%

Aqueous Solubility Assay

As shown in the table below, the aqueous solubility of integrin alpha-4-beta-7-inhibiting nacellins was relatively high, with a mean solubility of 715 microM. The range of solubilities measured in triplicate for 15 distinct compounds was 183 microM to greater than 1000 microM. Note that the maximum concentration evaluated was different for different test articles based on the presumed aqueous solubility. The compounds assessed include:

UM0131995-05 (Compound No. 4)

UM0132366-01 (Compound No. 87)

Attorney Docket No. 50412-119003

UM0132368-01 (Compound No. 88)

UM0132369-01 (Compound No. 89)

UM0132370-01 (Compound No. 52)

UM0132371-01 (Compound No. 90)

UM0132374-01 (Compound No. 65)

UM0132375-02 (Compound No. 42)

UM0132376-01 (Compound No. 92)

UM0132377-01 (an analog of Compound No. 92, in which the lysine residue has been replaced by an ornithine residue)

UM0134839-01 (an analog of Compound No. 455, in which the phenylalanine and betaHomoLys residues have been replaced by a tyrosine residue)

UM0134690-01 (Compound No. 358)

UM0134830-01 (an analog of Compound No. 159, in which the tyrosine and alanine residues have been exchanged with respect to position within the sequence)

UM0134677-01 (Compound No. 158)

UM0134700-01 (Compound No. 62)

Determined List of Maximal solubility Concentration (μM) RSD Compounds evaluated (μM) Rep 1 Rep 2 Rep 3 Mean SD (%) UM0131995-05 1000 883.9 897.3 914.7 898.6 15.4 1.7 UM0132366-01 500 474.2 477.4 451.9 467.8 13.9 3.0 UM0132368-01 1000 926.3 917.9 934.8 926.3 8.4 0.9 UM0132369-01 1000 874.4 885.9 887.3 882.5 7.1 0.8 UM0132370-01 200 187.0 182.4 180.2 183.2 3.5 1.9 UM0132371-01 1000 786.7 837.3 879.6 834.5 46.6 5.6 UM0132374-01 200 179.4 184.0 185.9 183.1 3.3 1.8 UM0132375-02 1000 966.6 960.0 936.8 954.5 15.6 1.6 UM0132376-01 1000 990.7 958.6 939.5 962.9 25.9 2.7 UM0132377-01 200 225.7 218.9 211.2 218.6 7.3 3.3 UM0134839-01 1000 1038.0 1000.1 1004.0 1014.0 20.8 2.1 UM0134690-01 1000 957.3 950.5 920.8 942.9 19.4 2.1 UM0134830-01 1000 1012.0 1016.4 971.7 1000.0 24.7 2.5 UM0134677-01 200 225.4 230.6 217.8 224.6 6.4 2.9 UM0134700-01 1000 1044.8 988.2 950.6 994.5 47.4 4.8

Cytochrome P450 Inhibition Assay

The inhibitory activity of various nacellins against four isoforms of cytochrome P450 was assessed using human liver microsomes. The four isoforms evaluated were: CYP2D5, CYP3A4, CYP2C9, and CYP1A2. As shown below, for the seven nacellins evaluated in this experiment, 85% of the results of the assays showed an IC₅₀>15 microM (above the limit of detection). However, IC₅₀s of <10 microM, and in one case, <1 microM, were recorded for a few compounds. These compounds were subjected to a structural analysis so as to understand the functional groups contributing to this mild CYP450-inhibiting activity. The compounds assessed include:

ET01792 (Compound No. 5)

ET00762-02 (an analog of Compound No. 5, in which the phenylalanine residue has been replaced by a tryptophan residue)

ET01813 (Compound No. 12)

ET00328-01 (an analog of Compound No. 4, in which the tyrosine, leucine, aspartic acid and threonine residues have been replaced by threonine, methyl leucine, valine and phenylalanine residues)

ET01827 (Compound No. 15)

ET01842-01 (Compound No. 413)

IC50 (mM) List of compounds CYP2D6 CYP3A4 CYP2C9 CYP1A2 ET01792-01 >15 >15 >15 >15 ET00762-02 >15 >15 >15 >15 ET01813-01 >15 >15 >15 >15 ET00328-01 >15 9.7 >15 >15 ET01827-01 >15 >15 >15 >15 ET01838-01 11 3.7 >15 >15 ET01842-01 >15 0.89* >15 >15 Quinidine 0.17 — — — Ketoconazole — 0.023 — — Miconazole — — 0.32 — α-naphthoflavone — — — 0.05

In Vivo T Lymphocyte Trafficking Analyses

The ability of several integrin alpha-4-beta-7-inhibiting nacellins to attenuate the trafficking of integrin alpha-4-beta-7-expressing T lymphocytes was demonstrated in in vivo pharmacodynamics studies in DSS-treated mice. As shown in FIG. 6, this study was conducted in mice exposed for 5 days to dextran sulfate in their drinking water. On day 6, single doses of test articles were administered and, 5-6 hours later, peripheral blood, mesenteric lymph nodes and other tissues were collected and assessed.

As shown, the murine anti-alpha-4-beta-7 monoclonal antibody (DATK32; 25 mg/kg) substantially reduces homing of integrin a4b7+ T lymphocytes to the mesenteric lymph nodes (“MLN”), but did not affect the counts of CD11a+ T lymphocytes in the peripheral blood. As for the nacellins, at 100 mg/kg, the high oral bioavailability nacellin, ET1792 (Compound No. 5), but not the low oral bioavailability nacellin, ET2154 (Compound No. 105), evoked a significant reduction of homing to the MLN following oral dosing. When the dose of ET2154 was increased to 200 mg/kg, it evoked a significant and robust attenuation of homing to the MLN. These results demonstrate the importance of oral bioavailability (and the concomitant systemic exposure) to attenuate trafficking of T lymphocytes via an integrin alpha-4-beta-7—MAdCAM facilitated extravasation event from high endothelial venules in gut-associated lymphoid tissues. Under no circumstances did test nacellins produce a significant change in the content of CD11a+ T cells in peripheral blood (similarly, no changes in the CD34 and CD45 T lymphocyte content in peripheral blood was recorded for any nacellin; data not shown).

In Vivo Pharmacokinetic Assessments in Rodents

We assessed the pharmacokinetic profile of several integrin alpha-4-beta-7-inhibiting nacellins following oral doses, and in some cases, intravenous doses for naïve mice.

As shown in the FIG. 7 below, one and two oral doses of ET1792 (Compound No. 5) at 48 mg/kg to naïve mice produced significant absorption and systemic exposure. The exposure (AUC-0-tlast) following the first dose was 1,475 h*ng/ml, whereas following the second dose, the exposure was 2,188 h*ng/ml. The maximum plasma concentration recorded after the first dose was nearly 1,600 ng/ml.

Referring to FIG. 8, in a second study of orally administered ET1792 (at 40 mg/kg), a similar profile was recorded, with an exposure (AUCO-tlast) of 589 h*ng/ml. The biphasic absorption and delayed Tmax is likely indicative of an initial transcellular perfusion of enterocytes followed by a more prolonged transcellular perfusion.

In a subsequent study of ET1813 (Compound No. 12), single doses were administered via oral gavage (40 mg/kg) and intravenous injection (5 mg/kg). The absorption of this compound was less than that of ET1792, with a calculated oral bioavailability of ˜1%, despite a significant terminal half-life of nearly 2 hours.

PK parameter summary for i.v. dosing. Parameter estimate for each animal Mean Parameter R01 R02 R03^(a) (n = 2) Sd C₀ (ng/mL) 6330 7590 9980 6960 n/a Apparent t_(1/2) (h) 1.45 2.79 0.225 2.12 n/a AUC_(0-inf) (h*ng/mL) 741 711 1290 726 n/a CL_(s) (mL/h/kg) 6740 7030 3880 6885 n/a MRT_(0-inf) (h) 0.253 0.136 0.166 0.195 n/a V_(ss) (mL/kg) 1700 957 645 1329 n/a

PK parameter summary for p.o. dosing Parameter estimate for each animal Parameter R04 R05 R06^(a) Mean Sd t_(max) (h) 0.0833 0.0833 0.0833 0.0833 0.000 C_(max) (ng/mL) 387 28.0 22.9 146 209 Apparent t_(1/2) (h) 4.47 0.638 0.466 1.86 2.26 AUC_(0-inf) (h*ng/mL) 166 3.77 3.74 57.8 93.7 MRT_(0-inf) (h) 1.48 0.449 0.409 0.779 0.607 F (%) 2.86 0.0649 0.0644 0.996 1.61

Another set of pharmacokinetic studies were performed on ET2451 (Compound No. 340) in naïve mice. In this study, compound exposure in plasma, colon and liver were measured following both single oral doses (40 mg/kg) and single intravenous doses (5 mg/kg). As illustrated in the FIG. 9 and in the tables below, ET2451 was found to have an oral bioavailability of approximately 11% in plasma, and closer to 100% in colon. Also noteworthy is the difference in elimination half-life: in plasma the half-life was found to be ˜30 minutes following oral dosing, whereas the half-life in colon was greater than 21 hours. We also assessed bioavailability in the liver (figure below), which was similar to that measured in the plasma (˜8%) but half-life was significantly longer at ˜8 hours following oral dosing. It is clear from this study and others that the main route of elimination of ET2451 and other nacellins is through hepatobiliary clearance.

Summary of Plasma PK Parameters for ETO2451 Estimate Parameter Unit i.v. p.o. C₀ ng/mL 1408 n/a t_(max) h 0.083 0.0833 C_(max) ng/mL 1326 4638 Apparent t_(1/2) h 2.00 0.537 AUC_(0-last) h*ng/mL 778 686 AUC_(0-inf) h*ng/mL 778 690 CL mL/kg/h 6426 n/a MRT_(0-inf) h 0.698 0.689 V_(ss) mL/kg 4487 n/a F % 100 11.0 n/a denotes not applicable.

Summary of Colon PK Parameters for ET02415 Estimate Parameter Unit i.v. p.o. t_(max) h 2.00 2.00 C_(max) ng/mL 8210 49115 Apparent t_(1/2) h 3.40 21.5 AUC_(0-last) h*ng/mL 17109 130746 AUC_(0-inf) h*ng/mL 17140 145327 MRT_(0-inf) h 3.43 4.59 AUC Ratio 22.0 211 (Colon/Plasma)

Summary of Liver PK Parameters for ET02451 Estimate Parameter Unit i.v. p.o. t_(max) h 0.0833 2.00 C_(max) ng/mL 16613 4792 Apparent t_(1/2) h 9.60 7.94 AUC_(0-last) h*ng/mL 8289 6024 AUC_(0-inf) h*ng/mL 8460 6054 MRT_(0-inf) h 2.00 2.11 AUC Ratio 10.9 8.78 (Liver/Plasma) C₀ Concentration extrapolated to time zero following an i.v. dose t_(max) time at which maximum concentration is observed C_(max) maximum observed concentration Apparent t_(1/2) apparent half-life AUC_(0-last) area under the concentration vs time curve from time 0 to the time of the last measurable concentration AUC_(0-inf) area under the concentration vs time curve from time 0 to infinity CL systemic clearance MRT_(0-tlast) mean residence time from time zero to the time of the last measurable concentration V_(ss) steady-state volume of distribution F oral bioavailability = (Dose^(iv) ^(·) AUC^(po))/(Dose^(po) ^(·) AUC^(iv)) ^(·) 100

8-Day Efficacy Study in DSS Model (Therapeutic)

We assessed the efficacy of two distinct nacellins in the DSS experimental model of ulcerative colitis: ET2451 (Compound No. 340) and ET2452 (Compound No. 341). As shown above, ET2451 demonstrates significant absorption from the gut and systemic exposure following oral dosing, whereas ET2452 is a low oral bioavailability entity. Both test compounds were administered b.i.d. to mice over the course of three days—following an initial 5-days exposure to DSS (2-3%) in their drinking water. The efficacy and pharmacodynamics effect of the nacellins was compared to the mouse anti-integrin alpha-4-beta-7 mAb, DATK32. ET2452 was administered both orally and, to another group, via i.p. injection. The objective of this experimental design was to demonstrate that, although it may not be efficacious when administered orally, it produced substantial efficacy with i.p. dosing.

Disease activity index (“DAI”) score was assessed individually based on the severity of three specific symptoms: blood in stool, stool consistency and general health assessment (posture, fur and dehydration), on Day 5 and 8. As shown in FIG. 10, DAI score increased significantly from Day 5 to Day 8 in DSS+vehicle control group. Oral administration of ET02451-01 and i.p. administration of DATK32 led to a reduction of 15% and 19% respectively, but only the ET02451-01-evoked effect proved statistically significant. Oral administration of ET02452-01 did not have any beneficial effect on DAI. In contrast, intraperitoneal ET02452-01 treatment led to a significant reduction of DAI score on Day 8, by 46% in comparison to DSS+vehicle control group (p<0.05). In fact, ET02452-01 i.p. treatment prevented the increased severity of UC symptoms observed in the control vehicle-treated group, from day 5 to day 8.

Ulcerative colitis is associated with inflammatory changes of the intestinal tract with reduction of the length of the mice colon (raw data in Annex IV). DSS+vehicle control group showed a mean colon length of 4.3±0.3 cm and a lesion length of 1.7±0.3 cm corresponding to a lesion/colon length of 40% (FIG. 11). Treatments did not have any effect on colon length (FIG. 11). However, ET02451-01 significantly reduced the lesion length of this group (p<0.05), leading to a significant improvement of the lesion/colon length ratio, by reaching a value of lesion/colon length of 12% (p<0.05). Oral and i.p. administration of ET02452-01 led to a reduction of lesion length by 53 % and 20% respectively, in comparison to the control vehicle treated-group, but these reductions were not statistically significant (FIG. 11B). Beside, DATK32, administered on Day 5 by i.p. route, led to a reduction, not statistically significant, of 45% in comparison to the control vehicle treated-group.

Whether treatments would have been compared in separate experiments, a Student t-test would have been used for statistical comparison of each test article treated-group with the control vehicle treated-group. In that case, by a separated Student t-test, a statistical significant effect by oral ET02452-01 and i.p. DATK32 would have been obtained on lesion length and lesion/colon length.

The final score of colon inflammation was calculated by multiplying macroscopic score×lesion/colon length ratio for each mouse. Referring to FIG. 12, the measurement of this parameter shows a significant reduction of lesion inflammation by the oral administration of ET02451-01 (by 77%) and ET02452-01 (by 76%) in comparison to the control vehicle-treated group. Intraperitoneal administration of ET02452-01 and DATK32 led to a reduction of 39% and 53% respectively, but this effect was not statistically significant.

Cell Populations

There are no statistical differences between the percentage of CD3+CD4+CD11a+ T cell populations in vehicle mice and compound-treated ones in the three tissues tested (see FIG. 13). In all tissues, the population of CD34+ cells is the same in vehicle- and -nacellin-treated mice. However, a significant increase (p<0.01) of CD34+ cells is observed in the mice receiving the anti-α4β7 antibody (DATK32). For the CD3+CD4+α4β7+ cell population, no difference was observed in blood neither in peripheral lymph nodes. However, a significant decrease of this population was observed in mesenteric lymph nodes in mice receiving ET02452 (Compound No. 341) i.p. or ET02451 (Compound No. 340) p.o. Intraperitoneal administration of DATK32 also significantly decreased the percentage of CD3+CD4+α4β7+ T lymphocytes.

It is of note that over 600 macrocycles were made that exhibited less activity than those summarized in Tables 1A, 1B and 1C. A selection of the macrocycles with less or little activity are summarized in Tables 2A, 2B and 2C.

Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.

TABLE 1A Compound No. R¹ R² R³ R⁴ R⁵ 1 H H CH2—S—Ph H C(O)—NH-tert-Butyl 2 H H CH2—S—Ph H C(O)—NH-tert-Butyl 3 H H CH2—S—Ph H C(O)—NH-tert-Butyl 4 H H CH₃ H C(O)—NH-tert-Butyl 5 H H CH₃ H C(O)—NH-tert-Butyl 6 H H CH₃ H C(O)—NH-tert-Butyl 7 H H CH₃ H C(O)—NH-tert-Butyl 8 H H CH₃ H C(O)—NH-tert-Butyl 9 H H CH₃ H C(O)—NH-tert-Butyl 10 H H CH₃ H C(O)—NH-tert-Butyl 11 H H CH₃ H C(O)—NH-tert-Butyl 12 H H CH₃ H C(O)—NH-tert-Butyl 13 H H CH₃ H C(O)—NH-tert-Butyl 14 H H CH₃ H C(O)—NH-tert-Butyl 15 H H CH₃ H C(O)—NH-tert-Butyl 16 H H CH₃ H C(O)—NH-tert-Butyl 17 H H CH₃ H C(O)—NH-tert-Butyl 18 H H CH₃ H C(O)—NH-tert-Butyl 19 H H CH₃ H C(O)—NH-tert-Butyl 20 H H CH₃ H C(O)—NH-tert-Butyl 21 H H CH₃ H C(O)—NH-tert-Butyl 22 H H CH₃ H C(O)—NH-tert-Butyl 23 H H CH₃ H C(O)—NH-tert-Butyl 24 H H CH₃ H C(O)—NH-tert-Butyl 25 H H CH₃ H C(O)—NH-tert-Butyl 26 H H CH₃ H C(O)—NH-tert-Butyl 27 H H CH₃ H C(O)—NH-tert-Butyl 28 H H CH₃ H C(O)—NH-tert-Butyl 29 H H CH₃ H C(O)—NH-tert-Butyl 30 H H CH₃ H C(O)—NH-tert-Butyl 31 H H CH₃ H C(O)—NH-tert-Butyl 32 H H CH₃ H C(O)—NH-tert-Butyl 33 H H CH₃ H C(O)—NH-tert-Butyl 34 H H CH₃ H C(O)—NH-tert-Butyl 35 H H CH₃ H C(O)—NH-tert-Butyl 36 H H CH₃ H C(O)—NH-tert-Butyl 37 H H CH₃ H C(O)—NH-tert-Butyl 38 H H CH₃ H C(O)—NH-tert-Butyl 39 H H CH₃ H C(O)—NH-tert-Butyl 40 H H CH₃ H C(O)—NH-tert-Butyl 41 H H CH₃ H C(O)—NH-tert-Butyl 42 H H CH₃ H C(O)—NH-tert-Butyl 43 H CH₃ H H C(O)—NH-tert-Butyl 44 H H CH₃ H C(O)—NH-tert-Butyl 45 H H CH₃ H C(O)—NH-tert-Butyl 46 H H CH₃ H C(O)—NH-tert-Butyl 47 H H CH₃ H C(O)—NH-tert-Butyl 48 H H CH₃ H C(O)—NH-tert-Butyl 49 H H CH₃ H C(O)—NH-tert-Butyl 50 H H CH₃ H C(O)—NH-tert-Butyl 51 H H CH₃ H C(O)—NH-tert-Butyl 52 H H CH₃ H C(O)—NH-tert-Butyl 53 H H CH₃ H C(O)—NH-tert-Butyl 54 H H CH₃ H C(O)—NH-tert-Butyl 55 H H CH₃ H C(O)—NH-tert-Butyl 56 H H CH₃ H C(O)—NH-tert-Butyl 57 H H CH₃ H C(O)—NH-tert-Butyl 58 H H CH₃ H C(O)—NH-tert-Butyl 59 H H CH₃ H C(O)—NH-tert-Butyl 60 H H CH₃ H C(O)—NH-tert-Butyl 61 H H CH₃ H C(O)—NH-tert-Butyl 52 H H CH₃ H C(O)—NH-tert-Butyl 53 H H CH₃ H C(O)—NH-tert-Butyl 54 H H CH₃ H C(O)—NH-tert-Butyl 65 H H CH₃ H C(O)—NH-tert-Butyl 66 H H CH₃ H C(O)—NH-tert-Butyl 67 H H CH₃ H C(O)—NH-tert-Butyl 68 H H CH₃ H C(O)—NH-tert-Butyl 69 H H CH₃ H C(O)—NH-tert-Butyl 70 H H CH₃ H C(O)—NH-tert-Butyl 71 H H CH₃ H C(O)—NH-tert-Butyl 72 H H CH₃ H C(O)—NH-tert-Butyl 73 H H CH₃ H C(O)—NH-tert-Butyl 74 H H CH₃ H C(O)—NH-tert-Butyl 75 H H CH₃ H C(O)—NH-tert-Butyl 76 H H CH₃ H C(O)—NH-tert-Butyl 77 H H CH₃ H C(O)—NH-tert-Butyl 78 H H CH₃ H C(O)—NH-tert-Butyl 79 H H CH₃ H C(O)—NH-tert-Butyl 80 H H CH₃ H C(O)—NH-tert-Butyl 81 H H CH₃ H C(O)—NH-tert-Butyl 82 H H CH₃ H C(O)—NH-tert-Butyl 83 H H CH₃ H C(O)—NH-tert-Butyl 84 H H CH₃ H C(O)—NH-tert-Butyl 85 H H CH₃ H C(O)—NH-tert-Butyl 86 H H CH₃ H C(O)—NH-tert-Butyl 87 H H CH₃ H C(O)—NH-tert-Butyl 88 H H CH₃ H C(O)—NH-tert-Butyl 89 H H CH₃ H C(O)—NH-tert-Butyl 90 H H CH₃ H C(O)—NH-tert-Butyl 91 H H CH₃ H C(O)—NH-tert-Butyl 92 H H CH₃ H C(O)—NH-tert-Butyl 93 H H CH₃ H C(O)—NH-tert-Butyl 94 H H CH₃ H C(O)—NH-tert-Butyl 95 H H CH₃ H C(O)—NH-tert-Butyl 96 H H CH₃ H C(O)—NH-tert-Butyl 97 H H CH₃ H C(O)—NH-tert-Butyl 98 H H CH₃ H C(O)—NH-tert-Butyl 99 H H CH₃ H C(O)—NH-tert-Butyl 100 H H CH₃ H C(O)—NH-tert-Butyl 101 H H CH₃ H C(O)—NH-tert-Butyl 102 H H CH₃ H C(O)—NH-tert-Butyl 103 H CH₃ H H C(O)—NH-tert-Butyl 104 H H CH₃ H C(O)—NH-tert-Butyl 105 H H CH₃ H C(O)—NH-tert-Butyl 106 H H CH₃ H C(O)—NH-tert-Butyl 107 H H CH₃ H C(O)—NH-tert-Butyl 108 H H CH₃ H C(O)—NH-tert-Butyl 109 H H CH₃ H C(O)—NH-tert-Butyl 110 H H CH₃ H C(O)—NH-tert-Butyl 111 H H CH₃ H C(O)—NH-tert-Butyl 112 H H CH₃ H C(O)—NH-tert-Butyl 113 H H CH₃ H C(O)—NH-tert-Butyl 114 H H CH₃ H C(O)—NH-tert-Butyl 115 H H CH₃ H C(O)—NH-tert-Butyl 116 H H CH₃ H C(O)—NH-tert-Butyl 117 H H CH₃ H C(O)—NH-tert-Butyl 118 H H CH₃ H C(O)—NH-tert-Butyl 119 H H CH₃ H C(O)—NH-tert-Butyl 120 H H CH₃ H C(O)—NH-tert-Butyl 121 H H CH₃ H C(O)—NH-tert-Butyl 122 H H CH₃ H C(O)—NH-tert-Butyl 123 H H CH₃ H C(O)—NH-tert-Butyl 124 H CH₃ H H C(O)—NH-tert-Butyl 125 H H CH₃ H C(O)—NH-tert-Butyl 126 H H CH₃ H C(O)—NH-tert-Butyl 127 H H CH₃ H C(O)—NH-tert-Butyl 128 H H CH₃ H C(O)—NH-tert-Butyl 129 H H CH₃ H C(O)—NH-tert-Butyl 130 H H CH₃ H C(O)—NH-tert-Butyl 131 H H CH₃ H C(O)—NH-tert-Butyl 132 H H CH₃ H C(O)—NH-tert-Butyl 133 H H CH₃ H C(O)—NH-tert-Butyl 134 H H CH₃ H C(O)—NH-tert-Butyl 135 H H CH₃ H C(O)—NH-tert-Butyl 136 H H CH₃ H C(O)—NH-tert-Butyl 137 H H CH₃ H C(O)—NH-tert-Butyl 138 H H CH₃ H C(O)—NH-tert-Butyl 139 H H CH₃ H C(O)—NH-tert-Butyl 140 H H CH₃ H C(O)—NH-tert-Butyl 141 H H CH₃ H C(O)—NH-tert-Butyl 142 H CH₃ H C(O)—NH-tert-Butyl H 143 H H CH₃ H C(O)—NH-tert-Butyl 144 H H CH₃ H C(O)—NH-tert-Butyl 145 H H CH₃ H C(O)—NH-tert-Butyl 146 H H CH₃ H C(O)—NH-tert-Butyl 147 H H CH₃ H C(O)—NH-tert-Butyl 148 H H CH₃ H C(O)—NH-tert-Butyl 149 H H CH₃ H C(O)—NH-tert-Butyl 150 H H CH₃ H C(O)—NH-tert-Butyl 151 H H CH₃ H C(O)—NH-tert-Butyl 152 H H CH₃ H C(O)—NH-tert-Butyl 153 H H CH₃ H C(O)—NH-tert-Butyl 154 H H CH₃ H C(O)—NH-tert-Butyl 155 H H CH₃ H C(O)—NH-tert-Butyl 156 H H CH₃ H C(O)—NH-tert-Butyl 157 H H CH₃ H C(O)—NH-tert-Butyl 158 H H CH₃ H C(O)—NH-tert-Butyl 159 H H CH₃ H C(O)—NH-tert-Butyl 160 H H CH₃ H C(O)—NH-tert-Butyl 161 H H CH₃ H C(O)—NH-tert-Butyl 162 H H CH₃ H C(O)—NH-tert-Butyl 163 H H CH₃ H C(O)—NH-tert-Butyl 164 H H CH₃ H C(O)—NH-tert-Butyl 165 H H CH₃ H C(O)—NH-tert-Butyl 166 H H CH₃ H C(O)—NH-tert-Butyl 167 H H CH₃ H C(O)—NH-tert-Butyl 168 H H CH₃ H C(O)—NH-tert-Butyl 169 H H CH₃ H C(O)—NH-tert-Butyl 170 H H CH₃ H C(O)—NH-tert-Butyl 171 H H CH₃ H C(O)—NH-tert-Butyl 172 H H CH₃ H C(O)—NH-tert-Butyl 173 H H CH₃ H C(O)—NH-tert-Butyl 174 H H CH₃ H C(O)—NH-tert-Butyl 175 H H CH₃ H C(O)—NH-tert-Butyl 176 H H CH₃ H C(O)—NH-tert-Butyl 177 H H CH₃ H C(O)—NH-tert-Butyl 178 H H CH₃ H C(O)—NH-tert-Butyl 179 H H CH₃ H C(O)—NH-tert-Butyl 180 H H CH₃ H C(O)—NH-tert-Butyl 181 H H CH₃ H C(O)—NH-tert-Butyl 182 H H CH₃ H C(O)—NH-tert-Butyl 183 H H CH₃ H C(O)—NH-tert-Butyl 184 H H CH₃ H C(O)—NH-tert-Butyl 185 H H CH₃ H C(O)—NH-tert-Butyl 186 H H CH₃ H C(O)—NH-tert-Butyl 187 H H CH₃ H C(O)—NH-tert-Butyl 188 H H CH₃ H C(O)—NH-tert-Butyl 189 H H CH₃ H C(O)—NH-tert-Butyl 190 H H CH₃ H C(O)—NH-tert-Butyl 191 H H CH₃ H C(O)—NH-tert-Butyl 192 H H CH₃ H C(O)—NH-tert-Butyl 193 H H CH₃ H C(O)—NH-tert-Butyl 194 H H CH₃ H C(O)—NH-tert-Butyl 195 H H CH₃ H C(O)—NH-tert-Butyl 196 H H CH₃ H C(O)—NH-tert-Butyl 197 H H CH₃ H C(O)—NH-tert-Butyl 198 H H CH₃ H C(O)—NH-tert-Butyl 199 H H CH₃ H C(O)—NH-tert-Butyl 200 H H CH₃ H C(O)—NH-tert-Butyl 201 H H CH₃ H C(O)—NH-tert-Butyl 202 H H CH₃ H C(O)—NH-tert-Butyl 203 H H CH₃ H C(O)—NH-tert-Butyl 204 H H CH₃ H C(O)—NH-tert-Butyl 205 H H CH₃ H C(O)—NH-tert-Butyl 206 H H CH₃ H C(O)—NH-tert-Butyl 207 H H CH₃ H C(O)—NH-tert-Butyl 208 H H CH₃ H C(O)—NH-tert-Butyl 209 H H CH₃ H C(O)—NH-tert-Butyl 210 H H CH₃ H C(O)—NH-tert-Butyl 211 H H CH₃ H C(O)—NH-tert-Butyl 212 H H CH₃ H C(O)—NH-tert-Butyl 213 H H CH₃ H C(O)—NH-tert-Butyl 214 H H CH₃ H C(O)—NH-tert-Butyl 215 H H CH₃ H C(O)—NH-tert-Butyl 216 H H CH₃ H C(O)—NH-tert-Butyl 217 H H CH₃ H C(O)—NH-tert-Butyl 218 H H CH₃ H C(O)—NH-tert-Butyl 219 H H CH₃ H C(O)—NH-tert-Butyl 220 H H CH₃ H C(O)—NH-tert-Butyl 221 H H CH₃ H C(O)—NH-tert-Butyl 222 H H CH₃ H C(O)—NH-tert-Butyl 223 H H CH₃ H C(O)—NH-tert-Butyl 224 H H CH₃ H C(O)—NH-tert-Butyl 225 H H CH₃ H C(O)—NH-tert-Butyl 226 H H CH₃ H C(O)—NH-tert-Butyl 227 H H CH₃ H C(O)—NH-tert-Butyl 228 H H CH₃ H C(O)—NH-tert-Butyl 229 H H CH₃ H C(O)—NH-tert-Butyl 230 H H CH₃ H C(O)—NH-tert-Butyl 231 H H CH₃ H C(O)—NH-tert-Buty) 232 H H CH₃ H C(O)—NH-tert-Butyl 233 H H CH₃ H C(O)—NH-tert-Butyl 234 H H CH₃ H C(O)—NH-tert-Butyl 235 H H CH₃ H C(O)—NH-tert-Butyl 236 H H CH₃ H C(O)—NH-tert-Butyl 237 H H CH₃ H C(O)—NH-tert-Butyl 238 H H CH₃ H C(O)—NH-tert-Butyl 239 H H CH₃ H C(O)—NH-tert-Butyl 240 H H CH₃ H C(O)—NH-tert-Butyl 241 H H CH₃ H C(O)—NH-tert-Butyl 242 H H CH₃ H C(O)—NH-tert-Butyl 243 H H CH₃ H C(O)—NH-tert-Butyl 244 H H CH₃ H C(O)—NH-tert-Butyl 245 H H CH₃ H C(O)—NH-tert-Butyl 246 H H CH₃ H C(O)—NH-tert-Butyl 247 H H CH₃ H C(O)—NH-tert-Butyl 248 H H CH₃ H C(O)—NH-tert-Butyl 249 H H CH₃ H C(O)—NH-tert-Butyl 250 H H CH₃ H C(O)—NH-tert-Butyl 251 H H CH₃ H C(O)—NH-tert-Butyl 252 H H CH₃ H C(O)—NH-tert-Butyl 253 H H CH₃ H C(O)—NH-tert-Butyl 254 H H CH₃ H C(O)—NH-tert-Butyl 255 H H CH₃ H C(O)—NH-tert-Butyl 256 H H CH₃ H C(O)—NH-tert-Butyl 257 H H CH₃ H C(O)—NH-tert-Butyl 258 H H CH₃ H C(O)—NH-tert-Butyl 259 H H CH₃ H C(O)—NH-tert-Butyl 260 H H CH₃ H C(O)—NH-tert-Butyl 251 H H CH₃ H C(O)—NH-tert-Butyl 262 H H CH₃ H C(O)—NH-tert-Butyl 263 H H CH₃ H C(O)—NH-tert-Butyl 264 H H CH₃ H C(O)—NH-tert-Butyl 265 H H CH₃ H C(O)—NH-tert-Butyl 266 H H CH₃ H C(O)—NH-tert-Butyl 267 H H CH₃ H C(O)—NH-tert-Butyl 268 H H CH₃ H C(O)—NH-tert-Butyl 269 H H CH₃ H C(O)—NH-tert-Butyl 270 H H CH₃ H C(O)—NH-tert-Butyl 271 H H CH₃ H C(O)—NH-tert-Butyl 272 H H CH₃ H C(O)—NH-tert-Butyl 273 H H CH₃ H C(O)—NH-tert-Butyl 274 H H CH₃ H C(O)—NH-tert-Butyl 275 H H CH₃ H C(O)—NH-tert-Butyl 275 H H CH₃ H C(O)—NH-tert-Butyl 277 H H CH₃ H C(O)—NH-tert-Butyl 278 H H CH₃ H C(O)—NH-tert-Butyl 279 H H CH₃ H C(O)—NH-tert-Butyl 280 H H CH₃ H C(O)—NH-tert-Butyl 281 H H CH₃ H C(O)—NH-tert-Butyl 282 H H CH₃ H C(O)—NH-tert-Butyl 283 H H CH₃ H C(O)—NH-tert-Butyl 284 H H CH₃ H C(O)—NH-tert-Butyl 285 H H CH₃ H C(O)—NH-tert-Butyl 286 H H CH₃ H C(O)—NH-tert-Butyl 287 H H CH₃ H C(O)—NH-tert-Butyl 288 H H CH₃ H C(O)—NH-tert-Butyl 289 H H CH₃ H C(O)—NH-tert-Butyl 290 H H CH₃ H C(O)—NH-tert-Butyl 291 H H CH₃ H C(O)—NH-tert-Butyl 292 H H CH₃ H C(O)—NH-tert-Butyl 293 H H CH₃ H C(O)—NH-tert-Butyl 294 H H CH₃ H C(O)—NH-tert-Butyl 295 PRO- PRO- H H C(O)—NH-tert-Butyl 296 H H CH₃ H C(O)—NH-tert-Butyl 297 H H CH₃ H C(O)—NH-tert-Butyl 298 H H CH₃ H C(O)—NH-tert-Butyl 299 H H CH₃ H C(O)—NH-tert-Butyl 300 H H CH₃ H C(O)—NH-tert-Butyl 301 H H CH₃ H C(O)—NH-tert-Butyl 302 H H CH₃ H C(O)—NH-tert-Butyl 303 H H CH₃ H C(O)—NH-tert-Butyl 304 H H CH₃ H C(O)—NH-tert-Butyl 305 H H CH₃ H C(O)—NH-tert-Butyl 306 H H CH₃ H C(O)—NH-tert-Butyl 307 H H CH₃ H C(O)—NH-tert-Butyl 308 H H CH₃ H C(O)—NH-tert-Butyl 309 H H CH₃ H C(O)—NH-tert-Butyl 310 H H CH₃ H C(O)—NH-tert-Butyl 311 H H CH₃ H C(O)—NH-tert-Butyl 312 H H CH₃ H C(O)—NH-tert-Butyl 313 H H CH₃ H C(O)—NH-tert-Butyl 314 H H CH₃ H C(O)—NH-tert-Butyl 315 H H CH₃ H C(O)—NH-tert-Butyl 316 H H CH₃ H C(O)—NH-tert-Butyl 317 H H CH₃ H C(O)—NH-tert-Butyl 318 H H CH₃ H C(O)—NH-tert-Butyl 319 H H CH₃ H C(O)—NH-tert-Butyl 320 H H CH₃ H C(O)—NH-tert-Butyl 321 H H CH₃ H C(O)—NH-tert-Butyl 322 H H CH₃ H C(O)—NH-tert-Butyl 323 H H CH₃ H C(O)—NH-tert-Butyl 324 H H CH₃ H C(O)—NH-tert-Butyl 325 H H CH₃ H C(O)—NH-tert-Butyl 326 H H CH₃ H C(O)—NH-tert-Butyl 327 H H CH₃ H C(O)—NH-tert-Butyl 328 H H CH₃ H C(O)—NH-tert-Butyl 329 H H CH₃ H C(O)—NH-tert-Butyl 330 H H CH₃ H C(O)—NH-tert-Butyl 331 H H CH₃ H C(O)—NH-tert-Butyl 332 H H CH₃ H C(O)—NH-tert-Butyl 333 H H CH₃ H C(O)—NH-tert-Butyl 334 H H CH₃ H C(O)—NH-tert-Butyl 335 H H CH₃ H C(O)—NH-tert-Butyl 335 H H CH₃ H C(O)—NH-tert-Butyl 337 H H CH₃ H C(O)—NH-tert-Butyl 338 H H CH₃ H C(O)—NH-tert-Butyl 339 H H CH₃ H C(O)—NH-tert-Butyl 340 H H CH₃ H C(O)—NH-tert-Butyl 341 H H CH₃ H C(O)—NH-tert-Butyl 342 H H CH₃ H C(O)—NH-tert-Butyl 343 H H CH₃ H C(O)—NH-tert-Butyl 344 H CH₃ H C(O)—NH-tert-Butyl H 345 H H CH₃ H C(O)—NH-tert-Butyl 346 H H CH₃ H C(O)—NH-tert-Butyl 347 H H CH₃ H C(O)—NH-tert-Butyl 348 H H CH₃ H C(O)—NH-tert-Butyl 349 H H CH₃ H C(O)—NH-tert-Butyl 350 H H CH₃ H C(O)—NH-tert-Butyl 351 H H CH₃ C(O)—NH-tert-Butyl H 352 H H CH₃ H C(O)—NH-tert-Butyl 353 H H CH₃ H C(O)—NH-tert-Butyl 354 H H CH₃ H C(O)—NH-tert-Butyl 355 H H CH₃ H C(O)—NH-tert-Butyl 356 H H CH₃ H C(O)—NH-tert-Butyl 357 H H CH₃ H C(O)—NH-tert-Butyl 358 H H CH₃ H C(O)—NH-tert-Butyl 359 H H CH₃ H C(O)—NH-tert-Butyl 360 H H CH₃ H C(O)—NH-tert-Butyl 361 H H CH₃ H C(O)—NH-tert-Butyl 362 H H CH₃ H C(O)—NH-tert-Butyl 363 H H CH₃ H C(O)—NH-tert-Butyl 364 H H CH₃ H C(O)—NH-tert-Butyl 365 H H CH₃ H C(O)—NH-tert-Butyl 366 H H CH₃ H C(O)—NH-tert-Butyl 367 H H CH₃ H C(O)—NH-tert-Butyl 368 H H CH₃ H C(O)—NH-tert-Butyl 369 H H CH₃ H C(O)—NH-tert-Butyl 370 H H CH₃ H C(O)—NH-tert-Butyl 371 H H CH₃ H C(O)—NH-tert-Butyl 372 H H CH₃ H C(O)—NH-tert-Butyl 373 H H CH₃ H C(O)—NH-tert-Butyl 374 H H CH₃ H C(O)—NH-tert-Butyl 375 H H CH₃ H C(O)—NH-tert-Butyl 376 H H CH₃ H C(O)—NH-tert-Butyl 377 H H CH₃ H C(O)—NH-tert-Butyl 378 H H CH₃ H C(O)—NH-tert-Butyl 379 H H CH₃ H C(O)—NH-tert-Butyl 380 H H CH₃ H C(O)—NH-tert-Butyl 381 H H CH₃ H C(O)—NH-tert-Butyl 382 H H CH₃ H C(O)—NH-tert-Butyl 383 H H CH₃ H C(O)—NH-tert-Butyl 384 H H CH₃ H C(O)—NH-tert-Butyl 385 H H CH₃ H C(O)—NH-tert-Butyl 386 H H CH₃ H C(O)—NH-tert-Butyl 387 H H CH₃ H C(O)—NH-tert-Butyl 388 H H CH₃ H C(O)—NH-tert-Butyl 389 H H CH₃ H C(O)—NH-tert-Butyl 390 H H CH₃ H C(O)—NH-tert-Butyl 391 H H CH₃ H C(O)—NH-tert-Butyl 392 H H CH₃ H C(O)—NH-tert-Butyl 393 H H CH₃ H C(O)—NH-tert-Butyl 394 H H CH₃ H C(O)—NH-tert-Butyl 395 H H CH₃ H C(O)—NH-tert-Butyl 395 H H CH₃ H C(O)—NH-tert-Butyl 397 H H CH₃ H C(O)—NH-tert-Butyl

TABLE 1B Com- Seq. pound ID. No. No. R⁶ R⁷ R⁸ X^(y) X^(z) X¹ X² X³ 1 1 PRO PRO H Y L D V 2 2 PRO PRO H H L D V 3 3 PRO PRO H Y L D T 4 3 PRO PRO H Y L D T 5 4 PRO PRO H F L D T 6 5 PRO PRO H HomoPhe L D T 7 6 PRO PRO H Cha L D T 8 7 PRO PRO H W L D I 9 8 PRO PRO H 1Nal L D T 10 9 PRO PRO H 2Nal L D T 11 10 PRO PRO H W L D Thr(OBn) 12 11 PRO PRO H Bip L D T 13 12 PRO PRO H Tyr(OPh) L D T 14 13 PRO PRO H 1Nal L D I 15 14 PRO PRO H 2Nal L D I 16 15 PRO PRO H 2Nal L D Thr(OBn) 17 16 [(4S)-fluoro-Pro] [(4S)-fluoro-Pro] H W L D T 18 17 PRO PRO H Bip L D Thr(OBn) 19 18 PRO PRO H Tyr(2-tolyl diaryl ether) L D T 20 19 PRO PRO H Tyr(4-CF3 diaryl ether) L D T 21 20 PRO PRO H Tyr(4-methoxy diaryl ether) L D T 22 21 PRO PRO H Tyr(4-fluoro diaryl ether) L D T 23 22 PRO PRO H Tyr(2-methoxy diaryl ether) L D T 24 23 PRO PRO H Tyr(3-methoxy diaryl ether) L D T 25 24 PRO PRO H Tyr(3-fluoro diaryl ether) L D T 26 25 PRO PRO H Tyr(3,4-difluoro diaryl ether) L D T 27 26 PRO PRO H Tyr(3-methyl diaryl ether) L D T 28 27 PRO PRO H Tyr(3,4-dimethyl diaryl ether) L D T 29 28 PRO PRO H Tyr(4-CO2Me diaryl ether) L D T 30 29 PRO PRO H Tyr(3-CO2Me diaryl ether) L D T 31 30 PRO PRO H Tyr(4-CO2H diaryl ether) L D T 32 31 HYP HYP H F L D T 33 32 PRO PRO H metaY(Opr) L D T 34 33 PRO PRO H Orn(benzamide) L D Thr(OBn) 35 34 PRO PRO H Orn(acetamide) L D Thr(OBn) 36 35 PRO PRO H Orn(methanesulfonamide) L D Thr(OBn) 37 36 PRO PRO H Orn(ethylcarbamate) L D Thr(OBn) 38 37 PRO PRO H Orn(pentyl amide) L D Thr(OBn) 39 38 PRO PRO H R L D T 40 39 PRO PRO H F L D Thr(OMe) 41 40 PRO PRO H F L D Thr(OEt) 42 41 PRO PRO H dTyr L D T 43 42 PRO PRO H dTic L D T 44 43 HYP HYP H [3-(3′-pyridyl)-Ala] L D T 45 44 [(4R)-fluoro-Pro] [(4R)-fluoro-Pro] H F L D T 46 45 [(4R)-fluoro-Pro] [(4R)-fluoro-Pro] H Bip L D T 47 46 [(4R)-fluoro-Pro] [(4R)-fluoro-Pro] H [3-(3′-pyridyl)-Ala] L D T 48 47 [(4R)-fluoro-Pro] [(4R)-fluoro-Pro] H Y L D T 49 48 [(4S)-fluoro-Pro] [(4S)-fluoro-Pro] H Y L D T 50 49 PRO PRO H dArg L D T 51 50 PRO PRO H dPip L D T 52 51 PRO PRO H [3-(4-thiazolyl)-Ala] L D T 53 52 PRO PRO H Y L D I 54 53 PRO PRO H (4-aza-Phe) L D T 55 54 PRO PRO H Y L D Pen 56 55 PRO PRO H (vinyl-Br-Leu) L D T 57 56 PRO PRO H Hyp(OBn) L D T 58 56 PRO PRO H Hyp(OBn) L D T 59 57 PRO PRO H Dap(Cbz) L D T 60 58 PRO PRO H His(Bn) L D T 61 59 PRO PRO H (4-amino-Phe) L D T 62 60 PRO PRO H (4-aza-dPhe) L D T 63 61 PRO PRO H Hyp L D T 64 62 PRO PRO H dTrp L D T 65 63 PRO PRO H M L D T 66 64 PRO PRO H dMet L D T 67 65 PRO PRO H (4-guanidino-Phe) L D T 68 66 PRO PRO H (3-aza-Phe) L D T 69 42 PRO PRO H dTic L D T 70 67 PRO PRO H (3-aza-dPhe) L D T 71 68 PRO PRO H Nva L D T 72 69 PRO PRO H dNle L D T 73 70 PRO PRO H dLys L D T 74 71 PRO PRO H dPro L D T 75 72 PRO PRO H dOrn L D T 76 73 PRO PRO H (3-benzothienyl-Ala) L D T 77 74 PRO PRO H dTyr(OAllyl) L D T 78 75 PRO PRO H dSer(OBn) L D T 79 76 PRO PRO H [3-(4-thiazolyl)-dAla] L D T 80 77 PRO PRO H (3-benzothienyl-dAla) L D T 81 78 PRO PRO H [3-(2-thienyl)-dAla L D T 82 79 PRO PRO H (4-aminomethyl-Phe) L D T 83 80 PRO PRO H dOrn(dimethyl) L D T 84 81 PRO PRO H (4-amino-dPhe) L D T 85 82 PRO PRO H (4-aminomethyl-dPhe) L D T 86 83 PRO PRO H dTyr(OBn) L D T 87 84 PRO PRO H P L D T 88 85 PRO PRO H cycloLeu L D T 89 86 PRO PRO H Aic L D T 90 87 PRO PRO H Tyr(OAllyl) L D T 91 88 PRO PRO H Chg L D T 92 89 PRO PRO H K L D T 93 90 PRO PRO H (2-aza-dPhe) L D T 94 91 PRO PRO H (2-aza-Phe) L D T 95 92 PRO PRO H [2-(2-pyridyl)-4-thiazolyl-Ala] L D T 96 93 PRO PRO H [2-(3-pyridyl)-4-thiazolyl-Ala] L D T 97 94 PRO PRO H [2-(4-pyridyl)-4-thiazolyl-Ala] L D T 98 95 PRO PRO H dTiq L D T 99 96 PRO PRO H [1-(S)-isoindoline-carboxylic acid] L D T 100 97 PRO PRO H Y dThr L D T 101 98 PRO PRO H Y P L D T 102 99 PRO PRO H Y dPro L D T 103 100 PRO PRO H Y Sar L D T 104 101 PRO PRO H Y cycloLeu L D T 105 100 PRO PRO H Y Sar L D T 106 102 PRO PRO H (3-iodo-Phe) Sar L D T 107 103 PRO PRO H (4-iodo-Phe) Sar L D T 108 104 PRO PRO H (3,3-diphenyl-Ala) Sar L D T 109 105 PRO PRO H F dLys L D T 110 106 PRO PRO H Bip dLys L D T 111 107 PRO PRO H [3-(4-thiazolyl)-Ala] dLys L D T 112 108 PRO PRO H (3,3-diphenyl-Ala) dLys L D T 113 109 PRO PRO H Y dLys L D I 114 110 PRO PRO H Y dArg L D T 115 111 PRO PRO H Y dSer L D T 116 112 PRO PRO H Bip Sar L D T 117 113 PRO PRO H 1Nal Sar L D T 118 114 PRO PRO H Y Pip L D T 119 115 PRO PRO H (2-iodo-Phe) Sar L D T 120 116 PRO PRO H 1Nal dLys L D T 121 117 PRO PRO H Y dLys L D MeThr 122 118 PRO PRO H F Sar L D T 123 119 PRO PRO H Y dTic L D T 124 99 PRO PRO H Y dPro L D T 125 120 PRO PRO H Y dPip L D T 126 121 PRO PRO H F dPro L D T 127 122 PRO PRO H (3,4-dimethoxy-Phe) dPro L D T 128 123 PRO PRO H (3,4,5-trifluoro-Phe) dPro L D T 129 124 PRO PRO H (3,5-dibromo-Tyr) dPro L D T 130 125 PRO PRO H F dPip L D T 131 126 PRO PRO H [3-(4-thiazolyl)-Ala] dPip L D T 132 127 PRO PRO H (4-aminomethyl-Phe) dPip L D T 133 128 PRO PRO H [2-iodo-Phe] dPip L D T 134 129 PRO PRO H (2-phenyl-Phe) dPip L D T 135 130 PRO PRO H [2-(2-methoxy-phenyl)-Phe] dPip L D T 136 131 PRO PRO H [2-(3-methoxy-phenyl)-Phe] dPip L D T 137 132 PRO PRO H [2-(4-methoxy-phenyl)-Phe] dPip L D T 138 133 PRO PRO H Bip dPip L D T 139 134 PRO PRO H Y Hyp L D T 140 135 PRO PRO H Y dHyp L D T 141 136 PRO PRO H Y (cis-dHyp) L D T 142 137 dPRO H dPRO dTyr dPip L D T 143 138 PRO PRO H 1Nal dPip L D T 144 139 PRO PRO H 2Nal dPip L D T 145 140 PRO PRO H (4-aminomethyl-Phe) dTic L D T 146 141 PRO PRO H (3-aminomethyl-Phe) dTic L D T 147 142 PRO PRO H (3-aminomethyl-dPhe) dTic L D T 148 143 PRO PRO H MeTyr dPip L D T 149 144 PRO PRO H Y dPip L D alloThr 150 145 PRO PRO H Y dPip tertbutylAla D T 151 146 PRO PRO H [3-(4-thiazolyl)-Ala] dHyp L D T 152 147 PRO PRO H (4-aminomethyl-Phe) dHyp L D T 153 148 PRO PRO H Y dPip L D I 154 149 PRO PRO H Y dMeLys L D I 155 150 PRO PRO H Y dNle L D T 156 151 PRO PRO H F dHyp L D T 157 152 PRO PRO H Y dMeArg L D T 158 153 PRO PRO H Y G L D T 159 154 PRO PRO H Y A L D T 160 155 PRO PRO H Y dAla L D T 161 156 PRO PRO H M G L D T 162 157 PRO PRO H Tyr(OAllyl) Sar L D T 163 158 PRO PRO H Tyr(OAllyl) G L D T 164 159 PRO PRO H [3-(4-thiazolyl)-Ala] Sar L D T 165 160 PRO PRO H (4-aminomethyl-Phe) G L D T 166 161 PRO PRO H Tyr(OAllyl) dVal L D T 167 162 PRO PRO H Tyr(OAllyl) dSer L D T 168 163 PRO PRO H Tyr(OAllyl) dAla L D T 169 164 PRO PRO H Tyr(OAllyl) P L D T 170 165 PRO PRO H Tyr(OAllyl) dPro L D T 171 166 PRO PRO H [3-(4-thiazolyl)-Ala] dVal L D T 172 167 PRO PRO H [3-(4-thiazolyl)-Ala] dSer L D T 173 168 PRO PRO H [3-(4-thiazolyl)-Ala] dAla L D T 174 169 PRO PRO H [3-(4-thiazolyl)-Ala] P L D T 175 170 PRO PRO H [3-(4-thiazolyl)-Ala] dPro L D T 176 171 PRO PRO H (4-aminomethyl-Phe) P L D T 177 172 PRO PRO H (4-aminomethyl-Phe) dPro L D T 178 173 PRO PRO H cycloLeu P L D T 179 174 PRO PRO H [2-(2-pyridyl)-4-thiazolyl-Ala] Sar L D T 180 175 PRO PRO H [2-(2-pyridyl)-4-thiazolyl-Ala] dPro L D T 181 176 PRO PRO H [2-(3-pyridyl)-4-thiazolyl-Ala] Sar L D T 182 177 PRO PRO H [2-(3-pyridyl)-4-thiazolyl-Ala] dPro L D T 183 178 PRO PRO H [2-(4-pyridyl)-4-thiazolyl-Ala] dPro L D T 184 179 PRO PRO H [3-(2-aminobenzyl-4-thiazolyl)-Ala] Sar L D T 185 180 PRO PRO H [2-(amino-benzyl)-4-thiazolyl-Ala] dPro L D T 186 181 PRO PRO H dTyr dPip L D I 187 182 PRO PRO H (2-aminomethyl-Phe) Aze L D T 188 183 PRO PRO H Y dPip L D Abu 189 184 PRO PRO H (3-aminomethyl-Phe) dTic L D Abu 190 185 PRO PRO H (2,4-dichloro-Phe) dPip L D T 191 186 PRO PRO H (3-phenyl-dPhe) dPip L D T 192 187 PRO PRO H [3-(5-quinolinyl)-dPhe] dPip L D T 193 188 PRO PRO H Y betaHomoLys L D T 194 189 PRO PRO H Y betaHomoPro L D T 195 190 PRO PRO H Y betaHomoLys L D T 196 191 PRO PRO H Y 2Abz L D T 197 192 PRO PRO H F betaHomoLys L D T 198 193 PRO PRO H [3-(4-thiazolyl)-Ala] betaHomoLys L D T 199 194 PRO PRO H (4-aminomethyl-Phe) betaHomoLys L D T 200 195 PRO PRO H Y betaHomoLys L D Thr(OBn) 201 196 PRO PRO H MeTyr dbetaHomoLys L D T 202 197 PRO PRO H 1Nal betaHomoLys L D T 203 198 PRO PRO H 2Nal betaHomoLys L D T 204 199 PRO PRO H Bip betaHomoLys L D T 205 200 PRO PRO H (2-iodo-Phe) betaHomoLys L D T 206 201 PRO PRO H [2-(2,5-dimethyl-isoxazole)-Phe] betaHomoLys L D T 207 202 PRO PRO H (2-phenyl-Phe) betaHomoLys L D T 208 202 PRO PRO H (2-phenyl-Phe) betaHomoLys L D T 209 203 PRO PRO H [(2-piperazinyl-2-Phenyl)-Phe] betaHomoLys L D T 210 204 PRO PRO H Cha betaHomoLys L D T 211 205 PRO PRO H W betaHomoLys L D T 212 206 PRO PRO H dTrp betaHomoLys L D T 213 207 PRO PRO H (3-aminomethyl-Phe) betaHomoLys L D T 214 208 PRO PRO H (4-aminomethyl-dPhe) betaHomoLys L D T 215 209 PRO PRO H (4-aminomethyl-Phe) betaHomoLys L D I 216 210 PRO PRO H Y dbetaHomoLys L D I 217 211 PRO PRO H dArg betaHomoLys L D T 218 212 PRO PRO H (4-aminomethyl-Phe)-reduced betaHomoLys L D T 219 213 PRO PRO H [3-(4-thiazolyl)-Ala] dbetaHomoLys L D I 220 214 PRO PRO H F dbetaHomoLys L D I 221 215 PRO PRO H [3-(4-thiazolyl)-Ala] MebetaHomoLys L D T 222 216 PRO PRO H (4-aminomethyl-Phe) MebetaHomoLys L D T 223 217 PRO PRO H [3-(4-thiazolyl)-Ala] betaHomoLys L D I 224 218 PRO PRO H Tic betaHomoLys L D T 225 219 PRO PRO H dTic betaHomoLys L D T 226 220 PRO PRO H dTic dbetaHomoLys L D T 227 221 PRO PRO H Y betaHomoIle L D T 228 222 PRO PRO H (4-aminomethyl-Phe) betaHomoPro L D T 229 223 PRO PRO H Y dbetaHomoPro L D T 230 224 PRO PRO H (4-aminomethyl-Phe) dbetaHomoPro L D T 231 225 PRO PRO H R betaHomoLys L D T 232 226 PRO PRO H F MebetaHomoLys L D T 233 227 PRO PRO H Phe-reduced betaHomoLys L D T 234 228 PRO PRO H (3-aminomethyl-dPhe) betaHomoLys L D T 235 229 PRO PRO H -[3-(1-piperazinyl)phenyl]-Phe]-betaHomol betaHomoLys L D T 236 230 PRO PRO H [3-(4-thiazolyl)-dAla] betaHomoLys L D T 237 231 PRO PRO H (2-bromo-Phe) betaHomoLys L D T 238 232 PRO PRO H (2-chloro-Phe) betaHomoLys L D T 239 233 PRO PRO H (2-fluoro-Phe) betaHomoLys L D T 240 234 PRO PRO H (2-CF3-Phe) betaHomoLys L D T 241 235 PRO PRO H (2,4-dichloro-Phe) betaHomoLys L D T 242 236 PRO PRO H (2-aminomethyl-Phe) betaHomoLys L D T 243 237 PRO PRO H [2-(4-quinolinyl)-Phe] betaHomoLys L D T 244 238 PRO PRO H [2-(5-quinolinyl)-Phe] betaHomoLys L D T 245 239 PRO PRO H [2-(3-quinolinyl)-Phe] betaHomoLys L D T 246 240 PRO PRO H dhomoPhe betaHomoLys L D T 247 241 PRO PRO H (2-iodo-dPhe) betaHomoLys L D T 248 242 PRO PRO H (2-phenyl-dPhe) betaHomoLys L D T 249 243 PRO PRO H [(2-piperazinyl-2-Phenyl)-dPhe] betaHomoLys L D T 250 244 PRO PRO H Y betaHomoLys L D I 251 245 PRO PRO H Y betaHomoLys L D V 252 246 PRO PRO H dTyr betaHomoLys L D I 253 247 PRO PRO H (4-aminomethyl-dPhe) betaHomoLys L D I 254 248 PRO PRO H (4-aminomethyl-Phe) betaHomoLys L D V 255 249 PRO PRO H (3-iodo-Phe) betaHomoLys L D T 256 250 PRO PRO H (3-phenyl-Phe) betaHomoLys L D T 257 251 PRO PRO H [3-(2-methoxy-phenyl)-Phe] betaHomoLys L D T 258 252 PRO PRO H [3-(2,6-dimethoxy-phenyl)-Phe] betaHomoLys L D T 259 253 PRO PRO H [3-(2-trifluoromethoxy-phenyl)-Phe] betaHomoLys L D T 260 254 PRO PRO H (4-iodo-Phe) betaHomoLys L D T 261 255 PRO PRO H [4-(2-methoxy-phenyl)-Phe] betaHomoLys L D T 262 256 PRO PRO H [4-(2-trifluoromethoxy-phenyl)-Phe] betaHomoLys L D T 263 257 PRO PRO H alphaMePhe betaHomoLys L D T 264 258 PRO PRO H MePhe betaHomoLys L D T 265 259 PRO PRO H [3-(2,6-dimethyl-phenyl)-Phe] betaHomoLys L D T 266 260 PRO PRO H [3-(quinolin-4-yl)-Phe] betaHomoLys L D T 267 261 PRO PRO H [3-(3,4-difluoro-phenyl)-Phe] betaHomoLys L D T 268 262 PRO PRO H [4-(2,6-dimethyl-phenyl)-Phe] betaHomoLys L D T 269 263 PRO PRO H [4-(2-chloro-6-methoxy-phenyl)-Phe] betaHomoLys L D T 270 264 PRO PRO H [3-(4-thiazolyl)-Ala]-reduced betaHomoLys L D T 271 265 PRO PRO H [2-[4-(1-piperazinyl)phenyl]-Phe] betaHomoLys L D T 272 266 PRO PRO H [2-(2,6-dimethylphenyl)-Phe] betaHomoLys L D T 273 267 PRO PRO H [2-(benzothiazol-5-yl)-Phe] betaHomoLys L D T 274 268 PRO PRO H HomoPhe betaHomoLys L D T 275 269 PRO PRO H (piperidine-4-amino-4-carboxylic acid) betaHomoLys L D T 276 270 PRO PRO H [2-(2,5-dimethyl-isoxazole)-dPhe] betaHomoLys L D T 277 271 PRO PRO H dTyr betaHomoLys L D V 278 272 PRO PRO H (4-aminomethyl-dPhe) betaHomoLys L D T 279 273 PRO PRO H [2-(2-chloro-6-methoxyphenyl)-Phe] betaHomoLys L D T 280 274 PRO PRO H 2Igl betaHomoLys L D T 281 275 PRO PRO H d2Igl betaHomoLys L D T 282 276 PRO PRO H Atc betaHomoLys L D T 283 277 PRO PRO H Y betaHomoLys L D alloIle 284 278 PRO PRO H dTyr betaHomoLys L D alloIle 285 279 PRO PRO H (4-aminomethyl-Phe) betaHomoLys L D alloIle 286 280 PRO PRO H [2-[2,5-Bis(trifluoromethyl)phenyl]-Phe] betaHomoLys L D T 287 281 PRO PRO H [2-[2,5-Bis(trifluoromethyl)phenyl]-Phe] betaHomoLys L D T 288 282 PRO PRO H Aic betaHomoLys L D T 289 283 PRO PRO H P betaHomoLys L D T 290 284 PRO PRO H dPro betaHomoLys L D T 291 285 PRO PRO H Pip betaHomoLys L D T 292 286 PRO PRO H [2-(3-Pyridyl)-Phe] betaHomoLys L D T 293 287 PRO PRO H [2-(4-Pyridyl)-Phe] betaHomoLys L D T 294 288 PRO PRO H [2-(3-bromo-2-Pyridyl)-Phe] betaHomoLys L D T 295 289 PRO PRO H Y dbetaHomoLys L D T 296 290 PRO PRO H (N-benzyl-Gly) betaHomoLys L D T 297 291 PRO PRO H [2-(2-bromo-3-Pyridyl)-Phe] betaHomoLys L D T 298 292 PRO PRO H [3-(2-chloro-6-methoxy-phenyl)-Phe] betaHomoLys L D T 299 293 PRO PRO H [3-(benzothiazol-5-yl)-Phe] betaHomoLys L D T 300 294 PRO PRO H (2-aminomethyl-Phe) MebetaHomoLys L D T 301 295 PRO PRO H (2-aminomethyl-dPhe) MebetaHomoLys L D T 302 296 PRO PRO H [3-(4-thiazolyl)-dAla] MebetaHomoLys L D T 303 297 PRO PRO H [2-(2-trifluoromethoxy-phenyl)-dPhe] MebetaHomoLys L D T 304 298 PRO PRO H Tic MebetaHomoLys L D T 305 299 PRO PRO H dTic MebetaHomoLys L D T 306 300 PRO PRO H [2-(5-quinolinyl)-dPhe] betaHomoLys L D T 307 301 PRO PRO H Y betaHomoLys L D alloThr 308 302 PRO PRO H Y MebetaHomoLys L D alloThr 309 303 PRO PRO H MeTyr MebetaHomoLys L D T 310 304 PRO PRO H MeTyr MebetaHomoLys L D alloThr 311 305 PRO PRO H MePhe MebetaHomoLys L D T 312 306 PRO PRO H (2-fluoro-Phe) MebetaHomoLys L D T 313 307 PRO PRO H (2-fluoro-MePhe) MebetaHomoLys L D T 314 308 PRO PRO H (2,4-dichloro-Phe) MebetaHomoLys L D T 315 309 PRO PRO H (2,4-dichloro-MePhe) MebetaHomoLys L D T 316 310 PRO PRO H (2-aminomethyl-MePhe) MebetaHomoLys L D T 317 311 PRO PRO H [3-(2,6-dimethoxy-phenyl)-dPhe] betaHomoLys L D T 318 312 PRO PRO H [3-(4-Quinolinyl)-dPhe] betaHomoLys L D T 319 313 PRO PRO H betaHomoLys Aze L D T 320 314 PRO PRO H (3-phenyl-dPhe) betaHomoLys L D T 321 315 PRO PRO H [3-(2-trifluoromethoxy-phenyl)-dPhe] betaHomoLys L D T 322 316 PRO PRO H [3-(2-methoxy-phenyl)-dPhe] betaHomoLys L D T 323 317 PRO PRO H [2-(5-quinolinyl)-MePhe] MebetaHomoLys L D T 324 318 PRO PRO H F betaHomoNle L D T 325 319 PRO PRO H F MebetaHomoLys(Me)2 L D T 326 320 PRO PRO H MePhe MebetaHomoLys(Me)2 L D T 327 321 PRO PRO H M MebetaHomoLys L D T 328 322 PRO PRO H Igl MebetaHomoLys L D T 329 323 PRO PRO H HomoPhe MebetaHomoLys L D T 330 324 PRO PRO H Hyp(OBn) MebetaHomoLys L D T 331 325 PRO PRO H (1,2-cis-ACHC) MebetaHomoLys L D T 332 326 PRO PRO H MeMet MebetaHomoLys L D T 333 327 PRO PRO H betaHomoLys betaHomoLys L D T 334 328 PRO PRO H BetaHomoPhe MebetaHomoLys L D T 335 329 PRO PRO H betahomoMet MebetaHomoLys L D T 336 330 PRO PRO H Y (3-aminomethyl-4-bromo-benzoic acid) L D T 337 331 PRO PRO H Y [3-aminomethyl-4-(4-aza-phenyl)-benzoic acid] L D T 338 332 PRO PRO H Y [3-aminomethyl-4-(2,5-dimethyl-isoxazole)- L D T benzoic acid] 339 333 PRO PRO H Y [3-aminomethyl-4-(3-aminomethyl-phenyl)- L D T benzoic acid] 340 334 PRO PRO H [3-aminomethyl-4-[4-(1-piperazinyl)-phenyl]-benzoic acid] L D T 341 335 PRO PRO H [3-aminomethyl-4-(4-quinoinyl)-benzoic acid] L D T 342 336 PRO PRO H (3-aminomethyl-4-bromo-benzoic acid) L D T 343 337 PRO PRO H [3-aminomethyl-4-(2,5-dimethyl-isoxazole)-benzoic acid] L D T 344 338 dPRO H dPRO [3-aminomethyl-4-(4-pyridyl)-benzoic acid] L D T 345 339 PRO PRO H [3-aminomethyl-(4-methylpyrazole-3-yl)-benzoic acid] L D T 346 340 PRO PRO H [3-aminomethyl-4-(3-quinolinyl)-benzoic acid] L D T 347 341 PRO PRO H [3-aminomethyl-4-(5-quinolinyl)-benzoic acid] L D T 348 342 PRO PRO H [3-aminomethyl-4-[2-(1-piperazinyl)phenyl]-benzoic acid] L D T 349 343 PRO PRO H [3-aminomethyl-4-[3-(1-piperazinyl)phenyl]-benzoic acid] L D T 350 344 PRO PRO H [3-aminomethyl-4-[2-(3-(piperidin-4-ylmethoxy)phenyl]-benzoic L D T acid] 351 345 PRO PRO H [3-aminomethyl-4-(4-pyridyl)-benzoic acid] L D T 352 346 PRO PRO H [3-aminomethyl-4-(4-pyridyl)-benzoic acid] L D Thr(OBn) 353 347 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)-benzoic acid] L D alloThr 354 348 PRO PRO H [3-aminomethyl-4-[4-(1-piperazinyl)phenyl]-benzoic acid] L D T 355 349 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)]-benzoic acid tertbutylAla D T 356 334 PRO PRO H [3-aminomethyl-4-[4-(1-piperazinyl-4-FITC)phenyl]- L D T benzoic acid] 357 350 PRO PRO H (N-benzyl-3-aminomethyl-benzoic acid) L D T 358 351 PRO PRO H (3-aminomethyl-benzoic acid L D T 359 352 PRO PRO H (3-aminomethyl-5-bromo-benzoic acid) L D T 360 353 PRO PRO H (3-aminomethyl-6-bromo-benzoic acid) L D T Com- Seq. pound ID. No. No. R⁶ R⁷ R⁸ X^(y) X² X¹ X² X³ 361 336 PRO PRO H (3-aminomethyl-4-bromo-benzoic acid) L D T 362 354 PRO PRO H [3-aminomethyl-5-(4-aza-phenyl)-benzoic acid] L D T 363 355 PRO PRO H [3-aminomethyl-4-(3-thiophenyl)-benzoic acid] L D T 364 356 PRO PRO H [3-aminomethyl-4-(4-N,N-dimethyl-carboxamide-phenyl)-benzoic L D T acid] 365 357 PRO PRO H [3-aminomethyl-4-(4-aza-phenyl)-benzoic acid] L D T 366 358 PRO PRO H [3-aminomethyl-4-(3-aza-phenyl)-benzoic acid] L D T 367 359 PRO PRO H [3-aminomethyl-4-(4-hydroxy-phenyl)-benzoic acid] L D T 368 360 PRO PRO H [3-aminomethyl-4-(5-(2,4-dimethyl)thiazole)-benzoic acid] L D T 369 361 PRO PRO H [3-aminomethyl-4-(3-N,N-dimethylaniline)-benzoic acid] L D T 370 362 PRO PRO H [3-aminomethyl-4-(2-fluoro-pyridyl)-benzoic acid] L D T 371 363 PRO PRO H [3-aminomethyl-4-(5-pyrimidinyl)-benzoic acid] L D T 372 364 PRO PRO H [3-aminomethyl-4-(3-N,N-dimethyl-diaryl ether)-benzoic acid] L D T 373 365 PRO PRO H [3-aminomethyl-4-(3-CF3-phenyl)-benzoic acid] L D T 374 366 PRO PRO H [3-aminomethyl-4-(2,5-dimethoxy-phenyl)-benzoic acid] L D T 375 367 PRO PRO H [3-aminomethyl-4-[(2,3,4-tri-methoxy)-phenyl-benzoic acid] L D T 376 368 PRO PRO H [3-aminomethyl-4-(4-carboxy)-phenyl-benzoic acid] L D T 377 369 PRO PRO H [3-aminomethyl-4-(piperonyl)-benzoic acid] L D T 378 370 PRO PRO H (3-aminomethyl-4-piperidiny-benzoic acid) L D T 379 371 PRO PRO H (3-aminomethyl-4-morpholinyl-benzoic acid) L D T 380 372 PRO PRO H [3-aminomethyl-4-(N,N-dimethyl)-benzoic acid] L D T 381 373 PRO PRO H [3-aminomethyl-4-(2-aminomethylphenyl)-benzoic acid] L D T 382 374 PRO PRO H [3-aminomethyl-4-(3-aminomethylphenyl)-benzoic acid] L D T 383 375 PRO PRO H [3-aminomethyl-4-(4-aminomethylphenyl)-benzoic acid] L D T 384 376 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)-benzoic acid] L D Abu 385 377 H Nva H [3-aminomethyl-4-(4-quinolinyl)-benzoic acid] L D T 386 334 PRO PRO H [3-aminomethyl-4-[4-(1-piperazinyl-4-AlexaFluor 647)phenyl]- L D T benzoic acid] 387 378 PRO PRO H (N-methyl-3-aminomethyl-benzoic acid) L D T 388 379 PRO PRO H [N-methyl-3-aminomethyl-4-(4-quinolinyl)-benzoic acid] L D T 389 380 PRO PRO H [2-(5-quinolinyl)-Phe]-reduced betaHomoLys L D T 390 334 PRO PRO H [3-aminomethyl-4-[4-(1-piperazinyl)-phenyl]-benzoic acid] L D T 391 334 PRO PRO H [3-aminomethyl-4-[4-(1-piperazinyl)-phenyl]-benzoic acid] L D T 392 334 PRO PRO H [3-aminomethyl-4-[4-(1-piperazinyl)-phenyl]-benzoic acid] L D T 393 31 HYP HYP H F L D T 394 31 HYP HYP H F L D T 395 31 HYP HYP H F L D T 396 31 HYP HYP H F L D T 397 31 HYP HYP H F L D T

TABLE 1C Compound a4b7 ELISA a4b1 ELISA Selectivity RPMI 8866 cell No. IC₅₀ (mM) IC₅₀ (mM) ELISA IC₅₀ (mM) 1 0.164 0.162 0.988 2 0.109 0.185 1.697 3 0.192 0.475 2.474 25.000 4 0.129 0.357 2.8 11.782 5 0.087 0.062 0.7 7.916 6 0.103 0.200 1.9 7 0.117 0.190 1.6 23.000 8 0.103 0.096 0.9 9 0.061 0.106 1.7 10 0.052 0.070 1.3 11 0.051 0.094 1.8 3.602 12 0.063 0.113 1.8 8.885 13 0.097 0.171 1.8 19.520 14 0.026 0.025 1.0 2.664 15 0.040 0.026 0.7 3.071 16 0.086 0.053 0.6 1.624 17 0.173 26.923 18 0.120 19 0.114 15.044 20 0.146 8.716 21 0.092 9.466 22 0.100 11.556 23 0.176 0.458 2.6 18.880 24 0.087 0.192 2.2 7.632 25 0.096 0.209 2.2 12.431 26 0.088 0.236 2.7 14.070 27 0.067 0.161 2.4 10.478 28 0.117 0.264 2.3 12.562 29 0.073 0.167 2.3 8.133 30 0.058 0.162 2.8 9.277 31 0.057 0.215 3.7 7.950 32 0.100 0.311 3.1 11.161 33 0.090 0.324 3.6 13.059 34 0.043 0.083 1.9 1.153 35 0.039 0.096 2.5 1.230 36 0.112 0.215 1.9 2.392 37 0.036 0.063 1.8 0.856 38 0.065 0.120 1.9 1.899 39 0.152 0.595 3.9 7.576 40 0.063 0.119 1.9 41 0.042 0.106 2.5 42 0.079 0.232 2.9 43 0.026 0.072 2.8 44 0.083 0.188 2.3 45 0.074 0.238 3.2 46 0.106 0.258 2.4 47 0.061 0.135 2.2 6.777 48 0.094 0.332 3.5 20.686 49 0.137 0.326 2.4 17.374 50 0.023 0.290 12.6 3.709 51 0.031 0.102 3.3 52 0.075 0.367 4.9 14.719 53 0.182 21.956 54 0.190 23.916 55 0.113 0.119 1.1 56 0.058 0.200 3.5 4.203 57 0.059 0.148 2.5 58 0.156 0.445 2.9 59 0.197 0.610 3.1 60 0.066 0.214 3.3 6.554 61 0.063 0.223 3.6 52 0.027 0.115 4.3 2.548 63 0.107 0.251 2.3 64 0.046 0.268 5.8 5.367 65 0.005 0.095 18.1 1.033 66 0.093 0.326 3.5 6.348 67 0.075 0.341 4.5 5.093 68 0.067 0.280 4.2 4.158 69 0.022 0.060 2.7 2.646 70 0.035 0.099 2.9 1.163 71 0.184 0.816 4.4 72 0.151 0.409 2.7 7.284 73 0.144 1.247 8.6 17.304 74 0.100 0.763 7.6 15.503 75 0.171 1.209 7.1 13.166 76 0.114 0.466 4.1 6.267 77 0.036 0.185 5.1 5.633 78 0.069 0.272 3.9 6.479 79 0.110 0.552 5.0 13.217 80 0.053 0.556 10.6 3.599 81 0.054 0.241 4.5 5.405 82 0.073 0.213 2.9 5.716 83 0.179 1.226 6.9 32.316 84 0.035 0.218 6.2 6.143 85 0.052 0.206 3.9 4.229 86 0.050 0.167 3.3 4.074 87 0.019 0.269 14.1 88 0.011 0.166 14.9 89 0.016 0.232 14.4 90 0.009 0.317 35.0 91 0.126 1.824 14.5 92 0.053 1.063 19.9 93 0.078 0.311 4.0 6.009 94 0.080 0.250 3.1 9.484 95 0.125 0.303 2.4 96 0.138 0.321 2.3 97 0.124 0.311 2.5 98 0.021 0.058 2.7 99 0.057 0.154 2.7 100 0.132 0.453 3.4 4.446 101 0.129 0.609 4.7 16.092 102 0.021 0.136 6.6 1.464 103 0.108 1.531 15.1 104 0.120 0.506 4.2 105 0.110 1.734 15.8 9.731 106 0.059 1.109 18.7 107 0.150 2.390 16.0 108 0.077 0.814 10.5 13.867 109 0.133 3.312 24.9 15.287 110 0.185 3.923 21.3 21.753 111 0.100 3.923 39.3 12.926 112 0.138 3.008 21.7 17.420 113 0.052 0.709 13.7 7.634 114 0.083 1.889 22.8 6.866 115 0.125 1.121 9.0 15.436 116 0.166 1.385 8.4 117 0.158 1.381 8.7 118 0.112 0.132 1.2 14.202 119 0.079 1.688 21.5 14.057 120 0.157 3.000 19.1 121 0.192 2.187 11.4 122 0.090 1.666 18.6 16.615 123 0.007 0.019 2.5 1.138 124 0.013 0.104 8.3 1.172 125 0.025 0.458 18.4 1.925 126 0.024 0.135 5.6 1.232 127 0.025 0.196 7.8 128 0.026 0.296 11.4 129 0.065 0.636 9.7 130 0.022 0.125 5.6 1.327 131 0.026 0.080 3.1 132 0.029 0.309 10.8 3.626 133 0.015 0.080 5.3 134 0.023 0.178 7.6 135 0.024 0.119 4.9 136 0.032 0.209 6.6 137 0.033 0.254 7.8 138 0.024 0.118 5.0 139 0.100 0.073 0.7 140 0.053 0.512 9.6 141 0.019 0.036 2.0 142 0.164 0.084 0.5 143 0.033 0.068 2.1 144 0.043 0.027 0.6 6.083 145 0.023 0.045 2.0 3.268 146 0.016 0.012 0.7 0.672 147 0.052 0.039 0.8 148 0.085 0.105 1.2 149 0.046 0.546 12.0 12.600 150 0.054 0.447 8.2 151 0.053 0.218 4.1 152 0.102 1.347 13.2 153 0.006 0.017 2.8 0.125 154 0.117 2.664 22.8 155 0.054 1.085 20.3 156 0.019 0.258 13.3 1.412 157 0.067 3.707 55.3 158 0.110 1.537 14.0 15.746 159 0.053 0.467 8.9 41.275 160 0.141 1.349 9.5 8.794 161 0.135 2.035 15.1 6.662 162 0.107 1.875 17.5 16.696 163 0.126 1.389 11.0 22.489 164 0.127 3.288 25.8 30.192 165 0.128 2.918 22.8 30.337 166 0.179 1.382 7.7 167 0.147 1.997 13.6 168 0.077 1.051 13.6 17.847 169 0.176 0.488 2.8 170 0.013 0.104 8.0 1.033 171 0.128 0.658 5.1 14.357 172 0.096 1.030 10.7 9.922 173 0.054 0.719 13.4 12.042 174 0.160 0.619 3.9 175 0.018 0.130 7.2 0.986 176 0.189 1.202 6.3 177 0.019 0.463 24.0 2.853 178 0.027 0.113 4.1 2.710 179 0.174 2.656 15.2 180 0.013 0.068 5.1 0.841 181 0.180 2.272 12.6 182 0.017 0.083 5.0 1.128 183 0.014 0.105 7.5 1.070 184 0.099 0.953 9.6 185 0.018 0.095 5.4 0.662 186 0.062 0.027 0.4 187 0.083 0.404 4.9 188 0.027 0.189 7.0 7.308 189 0.018 0.019 1.0 2.251 190 0.021 0.145 7.0 2.470 191 0.083 4.020 48.4 192 0.118 6.823 57.8 37.800 193 0.092 0.303 3.3 5.621 194 0.038 0.207 5.4 4.617 195 0.049 1.917 38.9 7.931 196 0.158 0.275 1.7 197 0.044 1.327 30.2 7.441 198 0.041 1.223 29.9 5.089 199 0.069 3.138 45.2 19.350 200 0.134 0.352 2.6 201 0.061 0.695 11.4 202 0.086 0.680 8.0 203 0.055 0.534 9.8 204 0.063 0.429 6.8 205 0.047 1.517 32.2 2.231 206 0.046 2.890 63.0 27.621 207 0.025 0.460 18.5 208 0.019 0.522 28.1 4.679 209 0.035 1.977 56.9 16.508 210 0.072 1.148 16.0 211 0.060 2.511 42.2 8.101 212 0.068 2.190 32.1 213 0.055 2.247 41.2 10.605 214 0.069 4.222 60.8 72.055 215 0.033 0.413 12.4 216 0.123 2.509 20.4 217 0.034 1.088 31.8 218 0.190 3.135 16.5 219 0.147 3.253 22.1 220 0.096 1.740 18.2 221 0.015 0.165 11.1 0.248 222 0.013 0.212 16.1 0.325 223 0.015 0.122 8.2 0.549 224 0.055 2.978 53.9 10.962 225 0.099 4.523 45.6 18.130 226 0.094 10.797 115.0 4.076 227 0.034 0.047 1.4 1.491 228 0.034 0.503 14.7 229 0.058 0.075 1.3 230 0.120 0.131 1.1 231 0.031 0.993 32.0 232 0.012 0.110 8.9 0.353 233 0.094 3.861 41.0 19.372 234 0.099 3.203 32.3 235 0.025 1.553 62.6 4.614 236 0.060 6.203 104.2 7.320 237 0.020 0.870 43.9 5.131 238 0.025 1.049 42.3 8.425 239 0.020 0.641 32.3 4.407 240 0.027 0.905 33.2 12.040 241 0.031 3.207 103.4 6.006 242 0.067 5.307 79.0 8.335 243 0.026 0.767 29.4 2.007 244 0.016 0.753 46.7 0.719 245 0.024 0.414 17.5 3.067 246 0.120 17.702 147.1 247 0.035 4.514 132.8 15.134 248 0.045 3.088 69.2 16.371 249 0.045 4.233 94.8 23.107 250 0.017 0.150 8.7 0.401 251 0.024 0.349 14.8 1.386 252 0.032 0.390 12.1 2.408 253 0.069 1.087 15.6 254 0.055 1.803 33.0 255 0.043 3.024 69.7 256 0.072 3.246 45.1 9.562 257 0.058 1.604 27.5 258 0.056 1.584 28.4 259 0.058 5.995 102.8 4.279 260 0.165 9.562 58.1 261 0.096 23.155 241.0 16.926 262 0.080 3.740 47.0 263 0.102 2.345 23.1 264 0.117 5.560 47.5 265 0.039 1.818 46.2 266 0.037 1.206 33.0 11.641 267 0.044 1.936 44.1 20.440 268 0.076 1.868 24.6 269 0.056 1.764 31.6 270 0.160 17.562 109.8 18.900 271 0.033 1.151 34.6 272 0.041 2.383 58.1 273 0.012 0.303 24.6 1.730 274 0.026 0.454 17.5 7.938 275 0.101 0.779 7.7 276 0.134 14.235 106.2 277 0.052 0.357 6.9 278 0.104 1.062 10.2 279 0.100 5.847 58.2 280 0.010 0.400 39.7 2.150 281 0.144 3.161 21.9 282 0.119 0.626 5.2 283 0.128 1.495 11.7 284 0.046 0.228 5.0 285 0.089 0.553 6.2 286 0.064 5.236 81.9 287 0.084 3.553 42.1 288 0.136 1.664 12.2 289 0.038 0.349 9.3 1.242 290 0.067 1.894 28.4 291 0.035 0.777 22.4 8.742 292 0.030 0.374 12.4 293 0.019 0.198 10.6 4.008 294 0.045 0.937 20.7 295 0.094 20.950 222.7 18.900 296 0.155 14.698 94.8 297 0.037 0.786 21.3 298 0.076 4.349 57.2 299 0.002 0.090 41.5 0.556 300 0.022 0.225 10.4 0.672 301 0.018 0.846 47.6 1.020 302 0.012 0.598 51.6 1.764 303 0.020 0.497 24.8 1.662 304 0.015 0.293 19.0 0.191 305 0.008 0.221 26.6 3.533 306 0.104 2.763 26.5 307 0.091 4.343 47.8 308 0.039 0.480 12.3 1.982 309 0.008 0.023 3.0 0.126 310 0.017 0.300 17.6 0.434 311 0.007 0.198 27.6 0.158 312 0.011 0.145 13.4 0.273 313 0.011 0.206 19.2 0.210 314 0.011 0.138 12.8 0.305 315 0.013 0.312 24.9 0.431 316 0.022 0.349 16.2 0.690 317 0.047 0.685 14.5 9.408 318 0.091 1.513 16.6 319 0.065 0.309 4.8 320 0.163 0.127 0.8 321 0.101 7.368 72.7 322 0.093 4.166 44.7 323 0.025 0.297 11.8 1.056 324 0.110 1.058 9.6 11.844 325 0.020 0.170 8.6 0.714 326 0.017 0.476 28.4 0.280 327 0.010 0.128 13.2 0.308 328 0.010 0.234 24.1 0.368 329 0.005 0.050 10.6 0.326 330 0.005 0.179 32.9 0.185 331 0.016 0.093 6.0 0.399 332 0.010 0.120 12.5 0.140 333 0.046 0.757 16.5 12.922 334 5.061 335 4.956 336 0.162 0.917 5.6 337 0.061 0.177 2.9 338 0.041 0.177 4.4 339 0.051 0.299 5.8 340 0.019 0.048 2.5 0.263 341 0.012 0.026 2.1 0.306 342 0.041 0.139 3.4 343 0.018 0.029 1.6 0.269 344 0.052 0.107 2.1 345 0.039 0.052 1.3 346 0.028 0.011 0.4 0.580 347 0.023 0.030 1.3 348 0.027 0.041 1.5 349 0.023 0.043 1.9 0.479 350 0.027 0.055 2.0 351 0.160 0.184 1.2 352 0.024 0.005 0.2 0.070 353 0.031 0.103 3.3 354 0.050 0.175 3.5 355 0.048 0.069 1.4 356 0.017 0.027 1.6 357 0.102 0.406 4.0 358 0.127 1.108 8.7 34.923 359 0.053 0.450 8.5 7.880 360 0.125 0.779 6.2 18.937 361 0.049 0.288 5.9 2.843 362 0.043 0.238 5.6 363 0.022 0.105 4.8 1.571 364 0.018 0.074 4.0 0.602 365 0.017 0.064 3.7 0.638 366 0.023 0.059 2.6 0.384 367 0.018 0.053 3.0 0.535 368 0.010 0.024 2.4 0.342 369 0.024 0.069 2.9 0.974 370 0.015 0.047 3.1 0.661 371 0.016 0.055 3.4 0.482 372 0.024 0.104 4.3 2.133 373 0.018 0.074 4.1 0.879 374 0.018 0.081 4.5 1.246 375 0.015 0.067 4.5 1.164 376 0.019 0.078 4.1 1.135 377 0.013 0.045 3.6 0.839 378 0.042 0.182 4.3 379 0.033 0.161 4.9 380 0.041 0.217 5.3 381 0.010 0.010 1.1 0.323 382 0.012 0.025 2.0 383 0.006 0.017 2.7 0.403 384 0.020 0.049 2.5 2.260 385 0.039 0.023 0.6 1.548 386 0.044 0.034 0.8 2.604 387 0.063 6.133 96.7 16.142 388 0.009 0.102 12.0 0.336 389 0.042 0.234 5.535 6.664 390 0.010 0.017 1.749 0.019 391 0.011 0.025 2.138 0.022 392 0.011 0.062 5.427 0.056 393 0.035 0.109 3.137 0.635 394 0.039 0.133 3.429 0.860 395 0.042 0.126 2.998 1.521 396 0.044 0.063 1.424 1.953 397 0.079 0.158 1.987 2.061

TABLE 2A Compound No. R¹ R² R³ R⁴ R⁵ 5 H H CH₃ H C(O)—NH-tert-Butyl 398 H H H H H 399 H H CH₃ H C(O)—NH2 4 H H CH₃ H C(O)—NH-tert-Butyl 400 H H H C(O)—NH-tert-Butyl H 401 H H H H C(O)—NH-tert-Butyl 402 H H CH₃ H H 403 H H CH₃ C(O)—NH-tert-Butyl H 404 H CH₃ H C(O)—NH-tert-Butyl H 405 H H CH₃ H C(O)—NH-tert-Butyl 406 H H CH₃ H C(O)—NH-tert-Butyl 407 H H CH₃ H C(O)—NH-tert-Butyl 408 H H CH₃ H C(O)—NH-tert-Butyl 409 H H CH₃ H C(O)—NH-tert-Butyl 410 H H CH₃ H C(O)—NH-tert-Butyl 411 H H CH₃ H C(O)—NH-tert-Butyl 412 H H CH₃ H C(O)—NH-tert-Butyl 413 H H CH₃ H C(O)—NH-tert-Butyl 414 H H CH₃ H C(O)—NH-tert-Butyl 415 H H CH₃ H C(O)—NH-tert-Butyl 416 H H CH₃ H C(O)—NH-tert-Butyl 417 H H CH₃ H C(O)—NH-tert-Butyl 418 H CH₃ H C(O)—NH-tert-Butyl H 419 H CH₃ H C(O)—NH-tert-Butyl H 420 H H CH₃ H C(O)—NH-tert-Butyl 125 H H CH₃ H C(O)—NH-tert-Butyl 421 H CH₃ H C(O)—NH-tert-Butyl H 422 H H CH₃ H C(O)—NH-tert-Butyl 423 H H CH₃ H C(O)—NH-tert-Butyl 424 H H CH₃ H C(O)—NH-tert-Butyl 425 H H CH₃ H C(O)—NH-tert-Butyl 426 H H CH₃ H C(O)—NH-tert-Butyl 427 H H CH₃ H C(O)—NH-tert-Butyl 428 H H CH₃ H C(O)—NH-tert-Butyl 429 H H CH₃ H C(O)—NH-tert-Butyl 430 H H CH₃ H C(O)—NH-tert-Butyl 431 H H CH₃ H C(O)—NH-tert-Butyl 432 PRO- PRO- H H C(O)—NH-tert-Butyl 341 H H CH₃ H C(O)—NH-tert-Butyl 433 H H CH₃ H C(O)—NH-tert-Butyl 434 H H CH₃ H C(O)—NH-tert-Butyl 435 H H CH₃ H C(O)—NH-tert-Butyl 436 PRO- PRO- H C(O)—NH-tert-Butyl H 437 H H CH₃ H C(O)—NH-tert-Butyl 232 H H CH₃ H C(O)—NH-tert-Butyl 438 H H CH₃ H C(O)—NH-tert-Butyl 439 H H CH₃ H C(O)—NH-tert-Butyl 440 H H CH₃ H C(O)—NH-tert-Butyl 441 H H CH₃ H C(O)—NH-tert-Butyl 442 H H CH₃ H C(O)—NH-tert-Butyl 443 H H CH₃ C(O)—NH-tert-Butyl H 444 H CH₃ H C(O)—NH-tert-Butyl H 445 PRO- PRO- H C(O)—NH-tert-Butyl H 446 PRO- PRO- H H C(O)—NH-tert-Butyl 447 H H CH₃ H C(O)—NH-tert-Butyl 448 H H CH₃ H C(O)—NH-tert-Butyl 449 H H CH₃ H C(O)—NH-tert-Butyl 450 H H CH₃ H C(O)—NH-tert-Butyl 270 H H CH₃ H C(O)—NH-tert-Butyl 451 H H CH₃ H C(O)—NH-tert-Butyl 452 H H CH₃ H C(O)—NH-tert-Butyl 453 H H CH₃ H C(O)—NH-tert-Butyl 454 H H CH₃ H C(O)—NH-tert-Butyl 455 H H CH₃ H C(O)—NH-tert-Butyl

TABLE 2B Com- Seq. pound ID. No. No. R⁶ R⁷ R⁸ X^(y) X^(z) X¹ X² X³ 5 4 PRO PRO H F L D T 398 4 PRO PRO H F L D T 399 4 PRO PRO H F L D T 4 3 PRO PRO H Y L D T 400 3 PRO PRO H Y L D T 401 3 PRO PRO H Y L D T 402 3 PRO PRO H Y L D T 403 3 PRO PRO H Y L D T 404 3 PRO PRO H Y L D T 405 381 PRO PRO H L D T Y 406 382 PRO PRO H D L D T 407 383 PRO PRO H 1-(R)-isoindoline-carboxylic acid L D T 408 384 PRO PRO H betaHomoLys L D T 409 385 PRO PRO H Y cyclopropylAla D T 410 386 PRO PRO H Y betaHomoLeu D T 411 387 PRO PRO H Y W D T 412 388 PRO PRO H Y L betaHomoAsp T 413 389 PRO PRO H 2Nal L HomoSer I 414 390 PRO PRO H 2Nal L Asp(ethyl ester) I 415 391 PRO PRO H Y L D H 416 392 PRO PRO H Y L D (2-aza-Phe) 417 393 PRO PRO H Y L D betaHomoThr 418 394 dPro H dPro dTyr dLeu dAsp dThr 419 395 dPro H dPro dThr dAsp dLeu dTyr 420 396 PRO PRO H dThr dAsp dLeu dTyr 125 120 PRO PRO H Y dPip L D T 421 397 dPro H dPro Y dPip L D T 422 398 PRO PRO H dTic G L D T 423 399 PRO PRO H dAla Y L D T 424 400 PRO PRO H (2-aminomethyl-phenylacetic acid) L D T 425 401 PRO PRO H Y dMet L D T 426 402 PRO PRO H Y dTiq L D T 427 403 PRO PRO H Y dPip MeLeu D T 428 404 PRO PRO H Y Sar HomocycloLeu D T 429 405 PRO PRO H Y dLys L D dThr 430 406 PRO PRO H Y dPip L D betaHomoIle 431 407 PRO PRO H Y L D T dAla 432 120 PRO PRO H Y dPip L D T 341 335 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)- L D T benzoic acid] 433 408 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)- MeLeu D T benzoic acid] 434 409 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)- L MeAsp T benzoic acid] 435 410 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)- L D MeThr benzoic acid] 436 335 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)- L D T benzoic acid] 437 411 PRO PRO H [3-aminomethyl-4-(4-quinolinyl)- K L D T benzoic acid] 232 226 PRO PRO H F MebetaHomoLys L D T 438 412 PRO PRO H [4-(2,6-dimethoxy-phenyl)-Phe] betaHomoLys L D T 439 413 PRO PRO H Y 1,2-trans-ACHC L D T 440 414 PRO PRO H Y betaAla L D T 441 415 PRO PRO H Y (2-aminobenzoic acid) L D T 442 416 PRO PRO H [3-(4-thiazolyl)-Ala] dMebetaHomoLys L D T 443 190 PRO PRO H Y betaHomoLys L D T 444 417 dPro H dPro Y betaHomoLys L D T 445 190 PRO PRO H Y betaHomoLys L D T 446 190 PRO PRO H Y betaHomoLys L D T 447 418 PRO PRO H L D T F betaHomoLys 448 419 PRO PRO H Y L G D T 449 420 PRO PRO H L D T A P 450 421 PRO PRO H Y L D T A 270 264 PRO PRO H [3-(4-thiazolyl)-Ala]-reduced betaHomoLys L D T 451 422 PRO PRO H [3-(4-thiazolyl)-Ala] betaHomoLys-reduced L D T 452 423 PRO PRO H [3-(4-thiazolyl)-Ala] betaHomoLys Leu-reduced D T 453 424 PRO PRO H [3-(4-thiazolyl)-Ala] betaHomoLys L Asp-reduced T 454 425 PRO PRO H [3-(4-thiazolyl)-Ala] betaHomoLys L D Thr-reduced 455 426 PRO PRO H F betaHomoLys-reduced L D T

TABLE 2C Compound a4b7 ELISA a4b1 ELISA Selectivity RPMI 8866 cell No. IC₅₀ (mM) IC₅₀ (mM) ELISA IC₅₀ (mM) 5 0.087 0.062 0.7 7.916 398 39.605 38.211 399 47 4 0.129 0.357 2.8 11.782 400 7.348 4.904 401 1.27 3.843 402 6.45 2.842 403 6.45 2.842 404 6.45 2.842 405 40.313 406 3.832 407 3.664 3.727 408 3.22 19.216 409 3.025 410 3.817 411 40 412 7.633 413 27.726 26.923 414 50 26.923 415 45.263 416 56.579 49.128 417 6.515 418 15.5 28.42 419 15.5 28.42 420 3.116 3 125 0.025 0.458 18.4 1.925 421 0.462 0.726 422 1.273 2.122 423 10 424 1.0 425 1.575 3.717 426 3.665 3.727 427 1.04 1.2 428 3.665 1.012 429 3.116 3.00 430 2.933 8.69 431 10 432 4.33 2.905 341 0.012 0.026 2.1 0.306 433 7.904 37.5 434 3.746 29.919 435 0.231 0.342 436 4.82 13.377 437 5.305 7.302 232 0.012 0.110 8.9 0.353 438 0.941 21.709 439 1.579 1.902 440 1.833 9.679 441 0.994 4.095 442 2.281 4.701 443 7.442 28.424 444 7.566 28.424 445 10.492 1.123 446 10.541 26.869 447 8.109 8.859 448 50 449 6.494 4.658 450 0.400 2.007 270 0.160 17.562 109.8 18.900 451 41.17 86.84 452 2.009 110 453 2.251 36.125 454 8.274 43.01 455 20.512 96.78

TABLE S1 Compound No. LC-MS (m/z) Experimental Protocol 1 A, E, Fb, I, M 2 A, E, Fb, I, M 3 A, E, Fb, I, M 4 A, E, Fa, Jb, I, M 5 A, E, Fa, Jb, I, M 6 A, E, Fa, Jb, I, M 7 A, E, Fa, Jb, I, M 8 A, E, Fa, Jb, I, M 9 780.4 A, E, Fa, Jb, I, M 10 780.4 A, E, Fa, Jb, I, M 11 859.4 A, E, Fa, Jb, I, M 12 806.4 A, E, Fa, Jb, I, M 13 A, E, Fa, Jb, H, I, M 14 792.4 A, E, Fa, Jb, I, M 15 792.4 A, E, Fa, Jb, I, M 16 A, E, Fa, Jb, I, M 17 A, E, Fa, Jb, I, M 18 A, E, Fa, Jb, I, M 19 A, E, Fa, Jb, H, I, M 20 A, E, Fa, Jb, H, I, M 21 A, E, Fa, Jb, H, I, M 22 A, E, Fa, Jb, H, I, M 23 A, E, Fa, Jb, H, I, M 24 A, E, Fa, Jb, H, I, M 25 A, E, Fa, Jb, H, I, M 26 A, E, Fa, Jb, H, I, M 27 836.4 A, E, Fa, Jb, H, I, M 28 A, E, Fa, Jb, H, I, M 29 880.4 A, E, Fa, Jb, H, I, M 30 880.4 A, E, Fa, Jb, H, I, M 31 866.4 A, E, Fa, Jb, H, I, M 32 A, E, Fa, Jb, I, M 33 A, E, Fa, Jb, H, I, M 34 891.4 A, E, Fa, Jb, K, I, M 35 829.4 A, E, Fa, Jb, K, I, M 36 A, E, Fa, Jb, K, I, M 37 859.4 A, E, Fa, Jb, K, I, M 38 871.4 A, E, Fa, Jb, K, I, M 39 A, E, Fa, Jb, I, M 40 744.4 A, E, Fa, Jb, I, M 41 758.4 A, E, Fa, Jb, I, M 42 746.4 A, D, I, M 43 742.4 A, D, I, M 44 A, E, Fa, Jb, I, M 45 748.4 A, E, Fa, Jb, I, M 46 A, E, Fa, Jb, I, M 47 749.3 A, E, Fa, Jb, I, M 48 A, E, Fa, Jb, I, M 49 A, E, Fa, Jb, I, M 50 739.4 A, D, I, M 51 A, D, I, M 52 A, D, I, M 53 A, D, I, M 54 A, D, I, M 55 A, D, I, M 56 A, D, I, M 57 A, D, I, M 58 A, D, I, M 59 A, D, I, M 60 A, D, I, M 61 A, D, I, M 62 A, D, I, M 63 A, D, I, M 64 A, D, I, M 65 A, D, I, M 66 A, D, I, M 67 A, D, I, M 68 A, D, I, M 69 A, D, I, M 70 A, D, I, M 71 A, D, I, M 72 A, D, I, M 73 A, D, I, M 74 A, D, I, M 75 A, D, I, M 76 A, D, I, M 77 A, D, I, M 78 A, D, I, M 79 A, D, I, M 80 A, D, I, M 81 A, D, I, M 82 A, D, I, M 83 A, D, I, M 84 A, D, I, M 85 A, D, I, M 86 A, D, I, M 87 A, D, I, M 88 A, D, I, M 89 A, D, I, M 90 A, D, I, M 91 A, D, I, M 92 A, D, I, M 93 A, D, I, M 94 A, D, I, M 95 A, D, G, I, M 96 A, D, G, I, M 97 A, D, G, I, M 98 A, D, I, M 99 A, D, I, M 100 847.4 A, D, I, M 101 843.6 A, D, I, M 102 843.6 A, D, I, M 103 817.4 A, D, I, M 104 857.4 A, D, I, M 105 817.4 A, D, I, M 106 927.2 A, D, I, M 107 927.2 A, D, I, M 108 877.4 A, D, I, M 109 858.4 A, D, I, M 110 934.4 A, D, I, M 111 865.4 A, D, I, M 112 934.4 A, D, I, M 113 886.4 A, D, I, M 114 902.4 A, D, I, M 115 833.4 A, D, I, M 116 877.4 A, D, I, M 117 851.4 A, D, I, M 118 857.4 A, D, I, M 119 927.2 A, D, I, M 120 908.4 A, D, I, M 121 888.4 A, D, I, M 122 801.4 A, D, I, M 123 905.4 A, D, I, M 124 843.4 A, D, I, M 125 857.4 A, D, I, M 126 827.4 A, D, I, M 127 887.4 A, D, I, M 128 881.4 A, D, I, M 129 1001.2 A, D, I, M 130 A, D, I, M 131 A, D, I, M 132 A, D, I, M 133 A, D, I, M 134 A, D, G, I, M 135 A, D, G, I, M 136 A, D, G, I, M 137 A, D, G, I, M 138 A, D, I, M 139 A, D, I, M 140 A, D, I, M 141 A, D, I, M 142 A, D, I, M 143 891.1 A, D, I, M 144 891.1 A, D, I, M 145 918.1 A, D, I, M 146 918.1 A, D, I, M 147 918.1 A, D, I, M 148 871.1 A, D, I, M 149 857.1 A, D, I, M 150 871.1 A, D, I, M 151 850.0 A, D, I, M 152 872.1 A, D, I, M 153 869.2 A, D, I, M 154 900.2 A, D, I, M 155 859.4 A, D, I, M 156 843.4 A, D, I, M 157 916.2 A, D, I, M 158 A, D, I, M 159 A, D, I, M 160 A, D, I, M 161 A, D, I, M 162 A, D, I, M 163 A, D, I, M 164 A, D, I, M 165 A, D, I, M 166 A, D, I, M 167 A, D, I, M 168 A, D, I, M 169 A, D, I, M 170 A, D, I, M 171 A, D, I, M 172 A, D, I, M 173 A, D, I, M 174 A, D, I, M 175 A, D, I, M 176 A, D, I, M 177 A, D, I, M 178 A, D, I, M 179 A, D, G, I, M 180 A, D, G, I, M 181 A, D, G, I, M 182 A, D, G, I, M 183 A, D, G, I, M 184 A, D, G, I, M 185 A, D, G, I, M 186 869.3 A, D, I, M 187 842.4 A, D, I, M 188 842.1 A, D, I, M 189 902.1 A, D, I, M 190 909.1 A, D, I, M 191 948.4 A, D, G, I, M 192 968.4 A, D, G, I, M 193 861.4 A, D, I, M 194 857.4 A, D, I, M 195 888.4 A, D, I, M 196 702.4 A, D, I, M 197 A, D, I, M 198 A, D, I, M 199 A, D, I, M 200 A, D, I, M 201 A, D, I, M 202 A, D, I, M 203 A, D, I, M 204 A, D, I, M 205 A, D, I, M 206 967.2 A, D, G, I, M 207 948.2 A, D, G, I, M 208 948.2 A, D, G, I, M 209 A, D, G, I, M 210 A, D, I, M 211 A, D, I, M 212 A, D, I, M 213 A, D, I, M 214 A, D, I, M 215 A, D, I, M 216 900.4 A, D, I, M 217 A, D, I, M 218 A, C, D, I, M 219 A, D, I, M 220 A, D, I, M 221 893.1 A, B, D, I, M 222 915.2 A, B, D, I, M 223 891.1 A, D, I, M 224 A, D, I, M 225 A, D, I, M 226 A, D, I, M 227 A, D, I, M 228 A, D, I, M 229 857.4 A, D, I, M 230 870.4 A, D, I, M 231 A, D, I, M 232 886.2 A, B, D, I, M 233 A, C, D, I, M 234 A, D, I, M 235 A, D, G, I, M 236 A, D, I, M 237 952.0 A, D, I, M 238 A, D, I, M 239 890.2 A, D, I, M 240 A, D, I, M 241 A, D, I, M 242 A, D, I, M 243 A, D, G, I, M 244 999.3 A, D, G, I, M 245 A, D, G, I, M 246 A, D, I, M 247 A, D, I, M 248 A, D, G, I, M 249 A, D, G, I, M 250 900.2 A, D, I, M 251 886.1 A, D, I, M 252 A, D, I, M 253 A, D, I, M 254 A, D, I, M 255 998.4 A, D, I, M 256 948.6 A, D, G, I, M 257 978.6 A, D, G, I, M 258 1008.6 A, D, G, I, M 259 1032.6 A, D, G, I, M 260 998.4 A, D, I, M 261 978.4 A, D, G, I, M 262 1032.4 A, D, G, I, M 263 886.6 A, D, I, M 264 886.6 A, D, I, M 265 976.6 A, D, G, I, M 266 1000.6 A, D, G, I, M 267 984.5 A, D, G, I, M 268 976.6 A, D, G, I, M 269 1012.6 A, D, G, I, M 270 A, C, D, J, M 271 A, D, G, I, M 272 A, D, G, I, M 273 1005.3 A, D, G, I, M 274 A, D, I, M 275 A, D, I, M 276 A, D, G, I, M 277 A, D, I, M 278 A, D, I, M 279 A, D, I, M 280 898.1 A, D, I, M 281 A, D, I, M 282 A, D, I, M 283 A, D, I, M 284 A, D, I, M 285 A, D, I, M 286 A, D, G, I, M 287 A, D, G, I, M 288 A, D, I, M 289 A, D, I, M 290 A, D, I, M 291 A, D, I, M 292 A, D, G, I, M 293 949.2 A, D, G, I, M 294 A, D, G, I, M 295 A, D, I, M 296 A, D, I, M 297 A, D, G, I, M 298 1012.6 A, D, G, I, M 299 1005.4 A, D, G, I, M 300 915.2 A, B, D, I, M 301 915.2 A, B, D, I, M 302 893.2 A, B, D, I, M 303 1047.3 A, B, D, I, M 304 898.2 A, B, D, I, M 305 898.2 A, B, D, I, M 306 A, D, G, I, M 307 A, D, I, M 308 A, B, D, I, M 309 916.2 A, B, D, I, M 310 916.2 A, B, D, I, M 311 900.2 A, B, D, I, M 312 904.2 A, B, D, I, M 313 918.2 A, B, D, I, M 314 954.1 A, B, D, I, M 315 968.1 A, B, D, I, M 316 929.2 A, B, D, I, M 317 A, D, G, I, M 318 999.4 A, D, G, I, M 319 808.6 A, D, I, M 320 917.4 A, D, G, I, M 321 1032.4 A, D, G, I, M 322 978.5 A, D, G, I, M 323 A, B, D, G, I, M 324 A, D, I, M 325 914.3 A, B, D, I, M 326 928.2 A, B, D, I, M 327 870.2 A, B, D, I, M 328 912.2 A, B, D, I, M 329 900.4 A, B, D, I, M 330 942.5 A, B, D, I, M 331 852.5 A, B, D, I, M 332 884.6 A, B, D, I, M 333 867.2 A, D, I, M 334 900.4 A, B, D, I, M 335 884.5 A, B, D, I, M 336 A, D, I, M 337 A, D, G, I, M 338 A, D, G, I, M 339 A, D, G, I, M 340 A, D, G, I, M 341 A, D, G, I, M 342 A, D, I, M 343 A, D, G, I, M 344 A, D, G, I, M 345 A, D, G, I, M 346 A, D, G, I, M 347 A, D, G, I, M 348 A, D, G, I, M 349 A, D, G, I, M 350 A, D, G, I, M 351 A, D, G, I, M 352 A, D, G, I, M 353 A, D, G, I, M 354 A, D, G, I, M 355 A, D, G, I, M 356 A, D, G, I, M, L, M 357 A, D, I, M 358 A, D, I, M 359 A, D, I, M 360 A, D, I, M 361 A, D, I, M 362 A, D, G, I, M 363 A, D, G, I, M 364 A, D, G, I, M 365 A, D, G, I, M 366 A, D, G, I, M 367 A, D, G, I, M 368 A, D, G, I, M 369 A, D, G, I, M 370 A, D, G, I, M 371 A, D, G, I, M 372 A, D, G, I, M 373 A, D, G, I, M 374 A, D, G, I, M 375 A, D, G, I, M 376 A, D, G, I, M 377 A, D, G, I, M 378 A, D, G, I, M 379 A, D, G, I, M 380 A, D, G, I, M 381 A, D, G, I, M 382 A, D, G, I, M 383 A, D, G, I, M 384 A, D, G, I, M 385 A, D, G, I, M 386 A, D, G, I, M 387 A, B, D, I, M 388 A, B, D, G, I, M 389 985.2 A, C, D, G, I, M 398 A, D, I, M 399 A, D, I, M 400 A, D, I, M 401 A, D, I, M 402 A, D, I, M 403 A, D, I, M 404 A, D, I, M 405 A, E, Fa, Jb, I, M 406 A, E, Fa, Jb, I, M 407 A, D, I, M 408 A, D, I, M 409 A, D, I, M 410 A, D, I, M 411 A, E, Fa, Jb, I, M 412 A, D, I, M 413 A, E, Fa, Jb, I, M 414 A, E, Fa, Jb, I, M 415 A, D, I, M 416 A, D, I, M 417 A, D, I, M 418 A, D, I, M 419 A, D, I, M 420 A, D, I, M 421 A, D, I, M 422 A, D, I, M 423 A, D, I, M 424 A, D, I, M 425 A, D, I, M 426 A, D, I, M 427 A, D, I, M 428 A, D, I, M 429 A, D, I, M 430 A, D, I, M 431 A, D, I, M 432 A, D, I, M 433 A, D, G, I, M 434 A, D, G, I, M 435 A, D, G, I, M 436 A, D, G, I, M 437 A, D, G, I, M 438 A, D, G, I, M 439 A, D, I, M 440 A, D, I, M 441 A, D, I, M 442 A, B, D, I, M 443 A, D, I, M 444 A, D, I, M 445 A, D, I, M 446 A, D, I, M 447 A, D, I, M 448 A, D, I, M 449 A, D, I, M 450 A, D, I, M 451 A, C, D, I, M 452 A, C, D, I, M 453 A, C, D, I, M 454 A, C, D, I, M 455 A, C, D, I, M 

1. A compound of formula (I):

wherein R¹ is H; C₁-C₆ alkyl; aryl; heteroaryl; alkenyl; or heterocycle; all of which are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkyl, alkoxy, vinyl, alkenyl, alkynyl, formyl, haloalkyl, halide, aryl, heteroaryl, amide, acyl, ester, ether, thioether, thioalkoxy, phosphino, and —NR_(a)R_(b), wherein R_(a) and R_(b) are independently selected from C₁-C₆ alkyl, aryl or benzyl; and where the one or more substituents is not alkyl when R¹ is C₁-C₆ alkyl; R² and R³ are each independently an amino acid side chain of a proteinogenic or a non-proteinogenic alpha-amino acid, or R² and R³ are covalently linked to each other to form a ring; R⁴ and R⁵ are each independently H; C₁-C₆ alkyl; aryl; heteroaryl; alkenyl; heterocycle; acids of the formula —C(O)OH; esters of the formula —C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula —C(O)NR**R***, wherein R** and R*** are independently selected from H, alkyl and aryl; —CH₂C(O)R, wherein R is selected from —OH, C₁-C₆alkyl, aryl, —C₁-C₆ alkyl-aryl, or —NR_(a)R_(b), where R_(a) and R_(b) are independently selected from C₁-C₆ alkyl, aryl or benzyl; or —C(O)R_(c), wherein R_(c) is selected from C₁-C₆ alkyl, aryl or —C₁-C₆ alkyl-aryl; or —C₁-C₆ alkyl-OR_(d), wherein R_(d) is an OH group or a protecting group selected from the group consisting of formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, benzenesulfonyl, p-toluenesulfonyl, benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, alpha.-,alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, benzyl, triphenylmethyl, benzyloxymethyl, and trimethylsilyl; all of which are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkyl, alkoxy, vinyl, alkenyl, alkynyl, formyl, haloalkyl, halide, aryl, heteroaryl, amide, acyl, ester, ether, thioether, thioalkoxy, phosphino, and —NR_(a)R_(b), wherein R_(a) and R_(b) are independently selected from C₁-C₆ alkyl, aryl or benzyl; and where, when R⁴, R⁵, R, R_(a), R_(b) or R_(c) is C₁-C₆ alkyl, the one or more substituents is not alkyl at that position; and wherein the one or more substituents is not —NR_(a)R_(b) when R is —NR_(a)R_(b); or R² or R³ are covalently linked to R¹ to form a cyclic secondary amine, and/or to R⁴ or R⁵ to form a ring, or R⁴ and R⁵ are covalently linked to each other to form a ring; R⁶ is H, C₁-C₆ alkyl, benzyl, alkenyl, C₁-C₆ alkyloxy; aryl; heteroaryl; heterocycle; —C(O)R****, wherein R**** is independently selected from alkyl, aryl, heteroaryl, amino, aminoalkyl, aminoaryl, aminoheteroaryl, alkoxy, aryloxy, heteroaryloxy; —CH₂C(O)R; or —C(O)R_(c); all of which are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkyl, alkoxy, vinyl, alkenyl, alkynyl, formyl, haloalkyl, halide, aryl, heteroaryl, amide, acyl, ester, ether, thioether, thioalkoxy, phosphino, and —NR_(a)R_(b), wherein R_(a) and R_(b) are independently selected from C₁-C₆ alkyl, aryl or benzyl, and where the one or more substituents is not alkyl when R⁶ is C₁-C₆ alkyl, or R⁶ forms, along with R⁷ or R⁸, a cyclic side chain of a proteinogenic or a non-proteinogenic amino acid having, the N-terminus thereof being the N—R⁶, wherein the proteinogenic or a non-proteinogenic amino acid is optionally substituted with a substituent selected from the group consisting of hydroxyl, cyano, alkyl, alkoxy, vinyl, alkenyl, alkynyl, formyl, haloalkyl, halide, aryl, heteroaryl, amide, acyl, ester, ether, thioether, thioalkoxy, phosphino, and —NR_(a)R_(b), wherein R_(a) and R_(b) are independently selected from C₁-C₆ alkyl, aryl or benzyl; R⁷ and R⁸ are independently selected from the amino acid side chains of a proteinogenic or a non-proteinogenic alpha-amino acid having the N-terminus thereof being the N—R⁶, or R⁷ or R⁸ forms a cyclic side chain with R⁶; stereocenters 1*, 2* and 3*, where present, are each independently selected from R and S; n is 1, 2, 3, or 4 and where n is 2-4, each R⁷ and each R⁸ are independent of each other; and wherein Z is an amino terminus of an amino acid; —C═O— adjacent L is the carboxy terminus of an amino acid; and L along with Z and —C═O— is a peptide having the following formula:

wherein X^(y) and X^(z) are each independently a proteinogenic or non-proteinogenic amino acid, or X^(z) is absent; X¹ is Leucine or tert-butyl-Ala; X² is Asp; and X³ is Thr(OMe); or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R¹ is H.
 3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R² and R³ are H and CH₃ respectively or vice versa.
 4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R⁴ and R⁵ are each independently H or C(O)—NHR***, wherein R*** is H or a C₁-C₆ alkyl.
 5. The compound of claim 4 or a pharmaceutically acceptable salt thereof, wherein R*** is tert-butyl.
 6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R⁶ and either R⁷ or R⁸ form a ring resulting in a proline residue having N—R⁶ as its N-terminus.
 7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein n is
 1. 8. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X¹ is Leu.
 9. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X² is Asp.
 10. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X^(y) is Tyr.
 11. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X^(z) is absent.
 12. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X^(y) is F and X^(z) is absent.
 13. The compound of claim 1, being compound 40 or a pharmaceutically acceptable salt thereof.
 14. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof along with the pharmaceutically acceptable carrier.
 15. The pharmaceutical composition of claim 14, formulated for oral delivery.
 16. The pharmaceutical composition of claim 14, formulated for topical delivery.
 17. The pharmaceutical composition of claim 14, formulated for parenteral delivery.
 18. A method of treating inflammation or an autoimmune disease in a patient, comprising administering to the patient a therapeutically effective amount of the compound of claim
 1. 19. The method of claim 18, wherein the inflammation or an autoimmune disease is gastrointestinal.
 20. The method of claim 18, wherein the condition or disease is Inflammatory Bowel Disease (IBD); ulcerative colitis; Crohn's disease; Celiac disease; enteropathy associated with seronegative arthropathies; microscopic colitis; collagenous colitis; eosinophilic gastroenteritis; radiotherapy; chemotherapy; pouchitis resulting after proctocolectomy and ileoanal anastomosis; gastrointestinal cancer; pancreatitis; insulin-dependent diabetes mellitus; mastitis; cholecystitis; cholangitis; pericholangitis; chronic bronchitis; chronic sinusitis; asthma; primary sclerosing cholangitis; human immunodeficiency virus (HIV) infection in the GI tract; eosinophilic asthma; eosinophilic esophagitis; gastritis; colitis; microscopic colitis; graft versus host disease; colitis associated with radio- or chemo-therapy; colitis associated with disorders of innate immunity; leukocyte adhesion deficiency-1; chronic granulomatous disease; glycogen storage disease type 1 b; Hermansky-Pudlak syndrome; Chediak-Higashi syndrome; and Wiskott-Aldrich Syndrome; osteoporosis; arthritis; multiple sclerosis; chronic pain; weight gain; or depression.
 21. The method of claim 20, wherein the condition is an inflammatory bowel disease.
 22. The method of claim 21, wherein the inflammatory bowel disease is ulcerative colitis.
 23. The method of claim 21, wherein the inflammatory bowel disease is Crohn's disease.
 24. The method of claim 18, wherein the compound inhibits binding of α4β7 integrin to MAdCAM.
 25. The method of claim 18, wherein the patient is a human. 